Alkylated piperazine compounds

ABSTRACT

Alkylated piperazine compounds of Formula I are provided, including stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, useful for inhibiting Btk kinase, and for treating immune disorders such as inflammation mediated by Btk kinase. Methods of using compounds of Formula I for in vitro, in situ, and in vivo diagnosis, and treatment of such disorders in mammalian cells, or associated pathological conditions, are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This non-provisional application filed under 37 CFR §1.53(b), claims thebenefit under 35 USC §119(e) of U.S. Provisional Application Ser. No.61/555,395 filed on 3 Nov. 2011, which is incorporated by reference inentirety.

FIELD OF THE INVENTION

The invention relates generally to compounds for treating disordersmediated by Bruton's Tyrosine Kinase (Btk) including inflammation,immunological, and cancer, and more specifically to compounds whichinhibit Btk activity. The invention also relates to methods of using thecompounds for in vitro, in situ, and in vivo diagnosis or treatment ofmammalian cells, or associated pathological conditions.

BACKGROUND OF THE INVENTION

Protein kinases, the largest family of human enzymes, encompass wellover 500 proteins. Bruton's Tyrosine Kinase (Btk) is a member of the Tecfamily of tyrosine kinases, and is a regulator of early B-celldevelopment as well as mature B-cell activation, signaling, andsurvival.

B-cell signaling through the B-cell receptor (BCR) can lead to a widerange of biological outputs, which in turn depend on the developmentalstage of the B-cell. The magnitude and duration of BCR signals must beprecisely regulated. Aberrant BCR-mediated signaling can causedisregulated B-cell activation and/or the formation of pathogenicauto-antibodies leading to multiple autoimmune and/or inflammatorydiseases. Mutation of Btk in humans results in X-linkedagammaglobulinemia (XLA). This disease is associated with the impairedmaturation of B-cells, diminished immunoglobulin production, compromisedT-cell-independent immune responses and marked attenuation of thesustained calcium sign upon BCR stimulation. Evidence for the role ofBtk in allergic disorders and/or autoimmune disease and/or inflammatorydisease has been established in Btk-deficient mouse models. For example,in standard murine preclinical models of systemic lupus erythematosus(SLE), Btk deficiency has been shown to result in a marked ameliorationof disease progression. Moreover, Btk deficient mice can also beresistant to developing collagen-induced arthritis and can be lesssusceptible to Staphylococcus-induced arthritis. A large body ofevidence supports the role of B-cells and the humoral immune system inthe pathogenesis of autoimmune and/or inflammatory diseases.Protein-based therapeutics (such as Rituxan) developed to depleteB-cells, represent an approach to the treatment of a number ofautoimmune and/or inflammatory diseases. Because of Btk's role in B-cellactivation, inhibitors of Btk can be useful as inhibitors of B-cellmediated pathogenic activity (such as autoantibody production). Btk isalso expressed in osteoclasts, mast cells and monocytes and has beenshown to be important for the function of these cells. For example, Btkdeficiency in mice is associated with impaired IgE-mediated mast cellactivation (marked diminution of TNF-alpha and other inflammatorycytokine release), and Btk deficiency in humans is associated withgreatly reduced TNF-alpha production by activated monocytes.

Thus, inhibition of Btk activity can be useful for the treatment ofallergic disorders and/or autoimmune and/or inflammatory diseases suchas: SLE, rheumatoid arthritis, multiple vasculitides, idiopathicthrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis,and asthma (Di Paolo et al (2011) Nature Chem. Biol. 7(1):41-50; Liu etal (2011) Jour. of Pharm. and Exper. Ther. 338(1):154-163). In addition,Btk has been reported to play a role in apoptosis; thus, inhibition ofBtk activity can be useful for cancer, as well as the treatment ofB-cell lymphoma, leukemia, and other hematological malignancies.Moreover, given the role of Btk in osteoclast function, the inhibitionof Btk activity can be useful for the treatment of bone disorders suchas osteoporosis. Specific Btk inhibitors have been reported (Liu (2011)Drug Metab. and Disposition 39(10):1840-1849; U.S. Pat. No. 7,884,108,WO 2010/056875; U.S. Pat. No. 7,405,295; U.S. Pat. No. 7,393,848; WO2006/053121; U.S. Pat. No. 7,947,835; US 2008/0139557; U.S. Pat. No.7,838,523; US 2008/0125417; US 2011/0118233; PCT/US2011/050034“PYRIDINONES/PYRAZINONES, METHOD OF MAKING, AND METHOD OF USE THEREOF”,filed 31 Aug. 2011; PCT/US2011/050013 “PYRIDAZINONES, METHOD OF MAKING,AND METHOD OF USE THEREOF”, filed 31 Aug. 2011; U.S. Ser. No. 13/102,720“PYRIDONE AND AZA-PYRIDONE COMPOUNDS AND METHODS OF USE”, filed 6 May2011).

SUMMARY OF THE INVENTION

The invention relates generally to Formula I, alkylated piperazinepyridone compounds with Bruton's Tyrosine Kinase (Btk) modulatingactivity.

Formula I compounds have the structures:

including stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof. The various substituents are defined herein below.

One aspect of the invention is a pharmaceutical composition comprised ofa Formula I compound and a pharmaceutically acceptable carrier, glidant,diluent, or excipient. The pharmaceutical composition may furthercomprise a second therapeutic agent.

Another aspect of the invention is a process for making a pharmaceuticalcomposition which comprises combining a Formula I compound with apharmaceutically acceptable carrier.

The invention includes a method of treating a disease or disorder whichmethod comprises administering a therapeutically effective amount of aFormula I compound to a patient with a disease or disorder selected fromimmune disorders, cancer, cardiovascular disease, viral infection,inflammation, metabolism/endocrine function disorders and neurologicaldisorders, and mediated by Bruton's tyrosine kinase.

The invention includes a kit for treating a condition mediated byBruton's tyrosine kinase, comprising: a) a first pharmaceuticalcomposition comprising a Formula I compound; and b) instructions foruse.

The invention includes a Formula I compound for use as a medicament, andfor use in treating a disease or disorder selected from immunedisorders, cancer, cardiovascular disease, viral infection,inflammation, metabolism/endocrine function disorders and neurologicaldisorders, and mediated by Bruton's tyrosine kinase.

The invention includes use of a Formula I compound in the manufacture ofa medicament for the treatment of immune disorders, cancer,cardiovascular disease, viral infection, inflammation,metabolism/endocrine function disorders and neurological disorders, andwhere the medicament mediates Bruton's tyrosine kinase.

The invention includes methods of making a Formula I compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the preparation of(S)-2-(5-Fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1(2H)-one101, starting with intermediate 2,6-Dibromo-4-fluorobenzaldehyde 101a.

FIG. 2 shows the preparation of(S)-5-[5-fluoro-2-(hydroxymethyl)-3(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-4,5-diazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3-trien-6-one102, starting with intermediate(S)-[4-fluoro-2-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)-piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.02,7]trideca-1(9),2(7),3-trien-5-yl}phenyl]methylacetate 102a.

FIG. 3 shows the preparation of(2S)-10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridine-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]-phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-9-one103, starting with intermediate (E)-Ethyl3-(2-Chloro-4,4-dimethylcyclopent-1-enyl)acrylate 103a.

FIG. 4 a shows the preparation of2-(3-(5-(5-((2S,5R)-2,5-Dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one104, starting with intermediate(2R,5S)-tert-Butyl-2,5-dimethyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate104a.

FIG. 4 b shows the preparation of4-Fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 104o from 3,4,6,7,8,9-Hexahydropyrazino[1,2-a]indol-1(2H)-one104j.

FIG. 5 shows the preparation of(S)-2-(3-(5-(5-(2-Ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one105, starting with intermediate(S)-2-(5-(5-(2-Ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 105a.

FIG. 6 shows the preparation of(S)-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one106, starting with intermediate(S)-4-fluoro-2-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 106a.

FIG. 7 shows the preparation of(S)-2-(3-(5-(5-(3,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one107, starting with intermediate (S)-tert-Butyl2-Methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 107a.

FIG. 8 shows the preparation of(R)-2-(3-(5-(5-(3,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)-phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one108, starting with intermediate (R)-tert-Butyl2-Methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 108a

FIG. 9 shows the preparation of(R)-2-(3-(5-(5-(2,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one109, starting with intermediate (R)-tert-Butyl3-Methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 109a.

FIG. 10 shows the preparation of(S)-2-(3-(5-(5-(2,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one110, starting with intermediate(S)-5-Bromo-3-(5-(2,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one110a.

FIG. 11 shows the preparation of2-(5-Fluoro-3-(5-(5-(3-(fluoromethyl)-4-methylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one111, starting with intermediate 1-tert-Butyl 2-Methyl4-benzylpiperazine-1,2-dicarboxylate 111a.

FIG. 12 shows the preparation of2-(5-Fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(9-methyl-7-oxa-3,9-diaza-bicyclo[3.3.1]nonan-3-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one112, starting with intermediate N,N-Dibromobenzenesulfonamide 112a.

FIG. 13 shows the preparation of(S)-2-(7,7-Difluoro-1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-4-fluoro-6-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)benzylAcetate 113q starting with intermediate (3S)-tert-Butyl3-Methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 113a.

FIG. 14 shows the preparation of 113o from intermediate ethyl1H-pyrrole-2-carboxylate.

FIG. 15 shows the preparation of2-Bromo-6-{4,4-dimethyl-9-oxo-7-thia-10,11-diazatricyclo[6.4.0.0^(2,6)]dodeca-1(8),2(6),11-trien-10-yl}-4-fluorobenzaldehyde 131i from intermediate3-methylcyclopent-2-enone.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. The present invention is in no way limited to the methods andmaterials described. In the event that one or more of the incorporatedliterature, patents, and similar materials differs from or contradictsthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application controls.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. The nomenclature used in this Application is based on IUPACsystematic nomenclature, unless indicated otherwise.

DEFINITIONS

When indicating the number of substituents, the term “one or more”refers to the range from one substituent to the highest possible numberof substitution, i.e. replacement of one hydrogen up to replacement ofall hydrogens by substituents. The term “substituent” denotes an atom ora group of atoms replacing a hydrogen atom on the parent molecule. Theterm “substituted” denotes that a specified group bears one or moresubstituents. Where any group may carry multiple substituents and avariety of possible substituents is provided, the substituents areindependently selected and need not to be the same. The term“unsubstituted” means that the specified group bears no substituents.The term “optionally substituted” means that the specified group isunsubstituted or substituted by one or more substituents, independentlychosen from the group of possible substituents. When indicating thenumber of substituents, the term “one or more” means from onesubstituent to the highest possible number of substitution, i.e.replacement of one hydrogen up to replacement of all hydrogens bysubstituents.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. In anotherembodiment, an alkyl radical is one to eight carbon atoms (C₁-C₈), orone to six carbon atoms (C₁-C₆). Examples of alkyl groups include, butare not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl(n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, —CH(CH₃)₂),1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (1-Bu,i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂)_(,) 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkylene” as used herein refers to a saturated linear orbranched-chain divalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkylene radical may be optionallysubstituted independently with one or more substituents described below.In another embodiment, an alkylene radical is one to eight carbon atoms(C₁-C₈), or one to six carbon atoms (C₁-C₆). Examples of alkylene groupsinclude, but are not limited to, methylene (—CH₂—), ethylene (—CH₂CH₂—),propylene (—CH₂CH₂CH₂—), and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted independentlywith one or more substituents described herein, and includes radicalshaving “cis” and “trans” orientations, or alternatively, “E” and “Z”orientations. Examples include, but are not limited to, ethylenyl orvinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), and the like.

The term “alkenylene” refers to linear or branched-chain divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenylene radical may be optionally substituted substitutedindependently with one or more substituents described herein, andincludes radicals having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations. Examples include, but are notlimited to, ethylenylene or vinylene (—CH═CH—), allyl (—CH₂CH═CH—), andthe like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to eight carbon atoms (C₂-C₈) with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and the like.

The term “alkynylene” refers to a linear or branched divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp triple bond, whereinthe alkynylene radical may be optionally substituted independently withone or more substituents described herein. Examples include, but are notlimited to, ethynylene (—C≡C—), propynylene (propargylene, —CH₂C≡C—),and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, for example, as a bicyclo[4,5],[5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10ring atoms can be arranged as a bicyclo[5,6] or [6,6] system, or asbridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Spiro moieties are also included within the scopeof this definition. Examples of monocyclic carbocycles include, but arenot limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cyclounaecyl, cyclododecyl, and the like. Carbocyclyl groups areoptionally substituted independently with one or more substituentsdescribed herein.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

“Arylene” means a divalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of two hydrogen atom from a twocarbon atoms of a parent aromatic ring system. Some arylene groups arerepresented in the exemplary structures as “Ar”. Arylene includesbicyclic radicals comprising an aromatic ring fused to a saturated,partially unsaturated ring, or aromatic carbocyclic ring. Typicalarylene groups include, but are not limited to, radicals derived frombenzene (phenylene), substituted benzenes, naphthalene, anthracene,biphenylene, indenylene, indenylene, 1,2-dihydronaphthalene,1,2,3,4-tetrahydronaphthyl, and the like. Arylene groups are optionallysubstituted with one or more substituents described herein.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to about 20 ring atoms in which atleast one ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles aredescribed in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, morpholin-4-yl, piperidin-1-yl,piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one,pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl,azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl,[1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl,benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl,pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups areoptionally substituted independently with one or more substituentsdescribed herein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

The terms “treat” and “treatment” refer to therapeutic treatment,wherein the object is to slow down (lessen) an undesired physiologicalchange or disorder, such as the development or spread of arthritis orcancer. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those with the conditionor disorder.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats the particulardisease, condition, or disorder, (ii) attenuates, ameliorates, oreliminates one or more symptoms of the particular disease, condition, ordisorder, or (iii) prevents or delays the onset of one or more symptomsof the particular disease, condition, or disorder described herein. Inthe case of cancer, the therapeutically effective amount of the drug mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can be measured, for example, by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

“Inflammatory disorder” as used herein can refer to any disease,disorder, or syndrome in which an excessive or unregulated inflammatoryresponse leads to excessive inflammatory symptoms, host tissue damage,or loss of tissue function. “Inflammatory disorder” also refers to apathological state mediated by influx of leukocytes and/or neutrophilchemotaxis.

“Inflammation” as used herein refers to a localized, protective responseelicited by injury or destruction of tissues, which serves to destroy,dilute, or wall off (sequester) both the injurious agent and the injuredtissue. Inflammation is notably associated with influx of leukocytesand/or neutrophil chemotaxis. Inflammation can result from infectionwith pathogenic organisms and viruses and from noninfectious means suchas trauma or reperfusion following myocardial infarction or stroke,immune response to foreign antigen, and autoimmune responses.Accordingly, inflammatory disorders amenable to treatment with Formula Icompounds encompass disorders associated with reactions of the specificdefense system as well as with reactions of the nonspecific defensesystem.

“Specific defense system” refers to the component of the immune systemthat reacts to the presence of specific antigens. Examples ofinflammation resulting from a response of the specific defense systeminclude the classical response to foreign antigens, autoimmune diseases,and delayed type hypersensitivity response mediated by T-cells. Chronicinflammatory diseases, the rejection of solid transplanted tissue andorgans, e.g., kidney and bone marrow transplants, and graft versus hostdisease (GVHD), are further examples of inflammatory reactions of thespecific defense system.

The term “nonspecific defense system” as used herein refers toinflammatory disorders that are mediated by leukocytes that areincapable of immunological memory (e.g., granulocytes, and macrophages).Examples of inflammation that result, at least in part, from a reactionof the nonspecific defense system include inflammation associated withconditions such as adult (acute) respiratory distress syndrome (ARDS) ormultiple organ injury syndromes; reperfusion injury; acuteglomerulonephritis; reactive arthritis; dermatoses with acuteinflammatory components; acute purulent meningitis or other centralnervous system inflammatory disorders such as stroke; thermal injury;inflammatory bowel disease; granulocyte transfusion associatedsyndromes; and cytokine-induced toxicity.

“Autoimmune disease” as used herein refers to any group of disorders inwhich tissue injury is associated with humoral or cell-mediatedresponses to the body's own constituents.

“Allergic disease” as used herein refers to any symptoms, tissue damage,or loss of tissue function resulting from allergy. “Arthritic disease”as used herein refers to any disease that is characterized byinflammatory lesions of the joints attributable to a variety ofetiologies. “Dermatitis” as used herein refers to any of a large familyof diseases of the skin that are characterized by inflammation of theskin attributable to a variety of etiologies. “Transplant rejection” asused herein refers to any immune reaction directed against graftedtissue, such as organs or cells (e.g., bone marrow), characterized by aloss of function of the grafted and surrounding tissues, pain, swelling,leukocytosis, and thrombocytopenia. The therapeutic methods of thepresent invention include methods for the treatment of disordersassociated with inflammatory cell activation.

“Inflammatory cell activation” refers to the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatability antigens or cell adhesion molecules) ininflammatory cells (including but not limited to monocytes, macrophages,T lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclearleukocytes such as neutrophils, basophils, and eosinophils), mast cells,dendritic cells, Langerhans cells, and endothelial cells). It will beappreciated by persons skilled in the art that the activation of one ora combination of these phenotypes in these cells can contribute to theinitiation, perpetuation, or exacerbation of an inflammatory disorder.

The term “NSAID” is an acronym for “non-steroidal anti-inflammatorydrug” and is a therapeutic agent with analgesic, antipyretic (loweringan elevated body temperature and relieving pain without impairingconsciousness) and, in higher doses, with anti-inflammatory effects(reducing inflammation). The term “non-steroidal” is used to distinguishthese drugs from steroids, which (among a broad range of other effects)have a similar eicosanoid-depressing, anti-inflammatory action. Asanalgesics, NSAIDs are unusual in that they are non-narcotic. NSAIDsinclude aspirin, ibuprofen, and naproxen. NSAIDs are usually indicatedfor the treatment of acute or chronic conditions where pain andinflammation are present. NSAIDs are generally indicated for thesymptomatic relief of the following conditions: rheumatoid arthritis,osteoarthritis, inflammatory arthropathies (e.g. ankylosing spondylitis,psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea,metastatic bone pain, headache and migraine, postoperative pain,mild-to-moderate pain due to inflammation and tissue injury, pyrexia,ileus, and renal colic. Most NSAIDs act as non-selective inhibitors ofthe enzyme cyclooxygenase, inhibiting both the cyclooxygenase-1 (COX-1)and cyclooxygenase-2 (COX-2) isoenzymes. Cyclooxygenase catalyzes theformation of prostaglandins and thromboxane from arachidonic acid(itself derived from the cellular phospholipid bilayer by phospholipaseA₂). Prostaglandins act (among other things) as messenger molecules inthe process of inflammation. COX-2 inhibitors include celecoxib,etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, andvaldecoxib.

The terms “cancer” refers to or describe the physiological condition inmammals that is typically characterized by unregulated cell growth. A“tumor” comprises one or more cancerous cells. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinomaof the lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

“Hematological malignancies” (British spelling “Haematological”malignancies) are the types of cancer that affect blood, bone marrow,and lymph nodes. As the three are intimately connected through theimmune system, a disease affecting one of the three will often affectthe others as well: although lymphoma is a disease of the lymph nodes,it often spreads to the bone marrow, affecting the blood. Hematologicalmalignancies are malignant neoplasms (“cancer”), and they are generallytreated by specialists in hematology and/or oncology. In some centers“Hematology/oncology” is a single subspecialty of internal medicinewhile in others they are considered separate divisions (there are alsosurgical and radiation oncologists). Not all hematological disorders aremalignant (“cancerous”); these other blood conditions may also bemanaged by a hematologist. Hematological malignancies may derive fromeither of the two major blood cell lineages: myeloid and lymphoid celllines. The myeloid cell line normally produces granulocytes,erythrocytes, thrombocytes, macrophages and mast cells; the lymphoidcell line produces B, T, NK and plasma cells. Lymphomas, lymphocyticleukemias, and myeloma are from the lymphoid line, while acute andchronic myelogenous leukemia, myelodysplastic syndromes andmyeloproliferative diseases are myeloid in origin. Leukemias includeAcute lymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML),Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML),Acute monocytic leukemia (AMOL) and small lymphocytic lymphoma (SLL).Lymphomas include Hodgkin's lymphomas (all four subtypes) andNon-Hodgkin's lymphomas (all subtypes).

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Il),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the Btk inhibitors of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of compounds of theinvention, including compounds produced by a process comprisingcontacting a Formula I compound of this invention with a mammal for aperiod of time sufficient to yield a metabolic product thereof.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand 1 or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or 1 meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity. Enantiomers may be separated from a racemic mixture bya chiral separation method, such as supercritical fluid chromatography(SFC). Assignment of configuration at chiral centers in separatedenantiomers may be tentative, and depicted in Table 1 structures forillustrative purposes, while stereochemical determination awaits, suchas x-ray crystallographic data.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The term “pharmaceutically acceptable salts” denotes salts which are notbiologically or otherwise undesirable. Pharmaceutically acceptable saltsinclude both acid and base addition salts. The phrase “pharmaceuticallyacceptable” indicates that the substance or composition must becompatible chemically and/or toxicologically, with the other ingredientscomprising a formulation, and/or the mammal being treated therewith.

The term “pharmaceutically acceptable acid addition salt” denotes thosepharmaceutically acceptable salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,carbonic acid, phosphoric acid, and organic acids selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid,pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid,ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamicacid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonicacid “mesylate”, ethanesulfonic acid, p-toluenesulfonic acid, andsalicyclic acid.

The term “pharmaceutically acceptable base addition salt” denotes thosepharmaceutically acceptable salts formed with an organic or inorganicbase. Examples of acceptable inorganic bases include sodium, potassium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, andaluminum salts. Salts derived from pharmaceutically acceptable organicnontoxic bases includes salts of primary, secondary, and tertiaryamines, substituted amines including naturally occurring substitutedamines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, and polyamine resins

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine.

The term “EC₅₀” is the half maximal effective concentration” and denotesthe plasma concentration of a particular compound required for obtaining50% of the maximum of a particular effect in vivo.

The term “Ki” is the inhibition constant and denotes the absolutebinding affinity of a particular inhibitor to a receptor. It is measuredusing competition binding assays and is equal to the concentration wherethe particular inhibitor would occupy 50% of the receptors if nocompeting ligand (e.g. a radioligand) was present. Ki values can beconverted logarithmically to pKi values (−log Ki), in which highervalues indicate exponentially greater potency.

The term “IC₅₀” is the half maximal inhibitory concentration and denotesthe concentration of a particular compound required for obtaining 50%inhibition of a biological process in vitro. IC₅₀ values can beconverted logarithmically to pIC₅₀ values (−log IC₅₀), in which highervalues indicate exponentially greater potency. The IC₅₀ value is not anabsolute value but depends on experimental conditions e.g.concentrations employed, and can be converted to an absolute inhibitionconstant (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol.(1973) 22:3099). Other percent inhibition parameters, such as IC₇₀,IC₉₀, etc., may be calculated.

The terms “compound of this invention,” and “compounds of the presentinvention” and “compounds of Formula I” include compounds of Formulas Iand stereoisomers, geometric isomers, tautomers, solvates, metabolites,and pharmaceutically acceptable salts and prodrugs thereof.

Any formula or structure given herein, including Formula I compounds, isalso intended to represent hydrates, solvates, and polymorphs of suchcompounds, and mixtures thereof.

Any formula or structure given herein, including Formula I compounds, isalso intended to represent unlabeled forms as well as isotopicallylabeled forms of the compounds. Isotopically labeled compounds havestructures depicted by the formulas given herein except that one or moreatoms are replaced by an atom having a selected atomic mass or massnumber. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, and chlorine, such as, but not limited to 2H(deuterium, D), 3H (tritium), 11C, 13C, 14C, 15N, 18F, 31P, 32P, 35S,36C1, and 125I. Various isotopically labeled compounds of the presentinvention, for example those into which radioactive isotopes such as 3H,13C, and 14C are incorporated. Such isotopically labelled compounds maybe useful in metabolic studies, reaction kinetic studies, detection orimaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. Deuterium labelled or substituted therapeutic compounds of theinvention may have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism, and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements. An18F labeled compound may be useful for PET or SPECT studies.Isotopically labeled compounds of this invention and prodrugs thereofcan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. Further, substitution with heavierisotopes, particularly deuterium (i.e., 2H or D) may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements or animprovement in therapeutic index. It is understood that deuterium inthis context is regarded as a substituent in the compound of the formula(I). The concentration of such a heavier isotope, specificallydeuterium, may be defined by an isotopic enrichment factor. In thecompounds of this invention any atom not specifically designated as aparticular isotope is meant to represent any stable isotope of thatatom. Unless otherwise stated, when a position is designatedspecifically as “H” or “hydrogen”, the position is understood to havehydrogen at its natural abundance isotopic composition. Accordingly, inthe compounds of this invention any atom specifically designated as adeuterium (D) is meant to represent deuterium.

Alkylated Piperazine Compounds

The present invention provides alkylated piperazine compounds of FormulaI, and pharmaceutical formulations thereof, which are potentially usefulin the treatment of diseases, conditions and/or disorders modulated byBtk kinase

Exemplary embodiments of Formula I compounds include

or stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof, wherein:

R¹, R² and R³ are independently selected from H, F, Cl, —NH₂, —NHCH₃,—N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OH, and C₁-C₃ alkyl;

R⁴ is selected from H, F, Cl, CN, —CH₂OH, —CH(CH₃)OH, —C(CH₃)₂OH,—CH(CF₃)OH, —CH₂F, —CHF₂, —CH₂CHF₂, —CF₃, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHC(O)CH₃, —OH, —OCH₃, —OCH₂CH₃,—OCH₂CH₂OH, cyclopropyl, cyclopropylmethyl, 1-hydroxycyclopropyl,imidazolyl, pyrazolyl, 3-hydroxy-oxetan-3-yl, oxetan-3-yl, andazetidin-1-yl;

R⁵ is selected from —CH₃, —CH₂CH₃, —CH₂OH, —CH₂F, —CHF₂, —CF₃, —CN, and—CH₂CH₂OH;

or two R⁵ groups form a 3-, 4-, 5-, or 6-membered carbocyclic orheterocyclic ring;

or an R⁵ group and an R⁸ group form a 3-, 4-, 5-, or 6-memberedcarbocyclic or heterocyclic ring;

n is 1, 2, 3, or 4

R⁶ is selected from H, F, —CH₃, —CH₂CH₃, —CH₂CH₂OH, —NH₂, and —OH;

R⁷ is selected from the structures:

where the wavy line indicates the site of attachment;

R⁸ is selected from —CH₃, —S(O)₂CH₃, cyclopropyl, azetidin-3-yl,oxetan-3-yl, and morpholin-4-yl;

X¹ is CR⁹ or N, where R⁹ is selected from H, F, Cl, —CH₃, —CH₂CH₃,—CH₂CH₂OH, —NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, and —OCH₂CH₂OH;

X² is CR¹⁰ or N, where R¹⁰ is selected from H, —CH₃, —CH₂CH₃, and—CH₂CH₂OH; and

Y¹ and Y² are independently selected from CH and N, where Y¹ and Y² arenot each

N.

Exemplary embodiments of Formula I compounds include wherein X is CR⁹,and R⁹ is H.

Exemplary embodiments of Formula I compounds include wherein X is N.

Exemplary embodiments of Formula I compounds include wherein R⁴ is—CH₂OH.

Exemplary embodiments of Formula I compounds include wherein R² is F.

Exemplary embodiments of Formula I compounds include wherein R¹ and R³are H.

Exemplary embodiments of Formula I compounds include wherein R⁶ is CH₃.

The Formula I compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention.

In addition, the present invention embraces all diastereomers, includingcis-trans (geometric) and conformational isomers. For example, if aFormula I compound incorporates a double bond or a fused ring, the cis-and trans-forms, as well as mixtures thereof, are embraced within thescope of the invention.

In the structures shown herein, where the stereochemistry of anyparticular chiral atom is not specified, then all stereoisomers arecontemplated and included as the compounds of the invention. Wherestereochemistry is specified by a solid wedge or dashed linerepresenting a particular configuration, then that stereoisomer is sospecified and defined.

The compounds of the present invention may exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms.

The compounds of the present invention may also exist in differenttautomeric forms, and all such forms are embraced within the scope ofthe invention. The term “tautomer” or “tautomeric form” refers tostructural isomers of different energies which are interconvertible viaa low energy barrier. For example, proton tautomers (also known asprototropic tautomers) include interconversions via migration of aproton, such as keto-enol and imine-enamine isomerizations. Valencetautomers include interconversions by reorganization of some of thebonding electrons.

Biological Evaluation

The relative efficacies of Formula I compounds as inhibitors of anenzyme activity (or other biological activity) can be established bydetermining the concentrations at which each compound inhibits theactivity to a predefined extent and then comparing the results.Typically, the preferred determination is the concentration thatinhibits 50% of the activity in a biochemical assay, i.e., the 50%inhibitory concentration or “IC₅₀”. Determination of IC₅₀ values can beaccomplished using conventional techniques known in the art. In general,an IC₅₀ can be determined by measuring the activity of a given enzyme inthe presence of a range of concentrations of the inhibitor under study.The experimentally obtained values of enzyme activity then are plottedagainst the inhibitor concentrations used. The concentration of theinhibitor that shows 50% enzyme activity (as compared to the activity inthe absence of any inhibitor) is taken as the IC₅₀ value. Analogously,other inhibitory concentrations can be defined through appropriatedeterminations of activity. For example, in some settings it can bedesirable to establish a 90% inhibitory concentration, i.e., IC₉₀, etc.

Formula I compounds were tested by a standard biochemical Btk KinaseAssay (Example 901).

A general procedure for a standard cellular Btk Kinase Assay that can beused to test Formula I compounds is a Ramos Cell Btk Assay (Example902).

A standard cellular B-cell proliferation assay can be used to testFormula I compounds with B-cells purified from spleen of Balb/c mice(Example 903).

A standard T cell proliferation assay can be used to test Formula Icompounds with T-cells purified from spleen of Balb/c mice (Example904).

A CD86 Inhibition assay can be conducted on Formula I compounds for theinhibition of B cell activity using total mouse splenocytes purifiedfrom spleens of 8-16 week old Balb/c mice (Example 905).

A B-ALL Cell Survival Assay can be conducted on Formula I compounds tomeasure the number of viable B-ALL cells in culture (Example 906).

A CD69 Whole Blood Assay can be conducted on Formula I compounds todetermine the ability of compounds to inhibit the production of CD69 byB lymphocytes in human whole blood activated by crosslinking surface IgMwith goat F(ab′)2 anti-human IgM (Example 907). CD69 is a type II C-typelectin involved in lymphocyte migration and cytokine secretion. CD69expression represents one of the earliest available indicators ofleukocyte activation and its rapid induction occurs throughtranscriptional activation (Vazquez et al (2009) Jour. of ImmunologyPublished Oct. 19, 2009, doi:10.4049/jimmuno1.0900839).Concentration-dependent inhibition of antigen receptor stimulation byselective Btk inhibitors induces cell surface expression of thelymphocyte activation marker CD69 (Honigberg et al (2010) Proc. Natl.Acad. Sci. 107(29):13075-13080). Thus, CD69 inhibition by selective Btkinhibitors may be correlated with therapeutic efficacy of certain B-celldisorders. The CD69 Hu Blood FACS IC70 values are displayed forexemplary Formula I compounds in Tables 1 and 2.

The cytotoxic or cytostatic activity of Formula I exemplary compoundscan be measured by: establishing a proliferating mammalian tumor cellline in a cell culture medium, adding a Formula I compound, culturingthe cells for a period from about 6 hours to about 5 days; and measuringcell viability (Example 908). Cell-based in vitro assays are used tomeasure viability, i.e. proliferation (IC₅₀), cytotoxicity (EC₅₀), andinduction of apoptosis (caspase activation) and may be useful inpredicting clinical efficacy against hematological malignancies andsolid tumors.

The in vitro potency of the combinations of Formula I compounds withchemotherapeutic agents can be measured by the cell proliferation assayof Example 908; the CellTiter-Glo® Luminescent Cell Viability Assay,commercially available from Promega Corp., Madison, Wis. Thishomogeneous assay method is based on the recombinant expression ofColeoptera luciferase (U.S. Pat. No. 5,583,024; U.S. Pat. No. 5,674,713;U.S. Pat. No. 5,700,670) and determines the number of viable cells inculture based on quantitation of the ATP present, an indicator ofmetabolically active cells (Crouch et al (1993) J. Immunol. Meth.160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay wasconducted in 96 or 384 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay can be used with various multiwellformats, e.g. 96 or 384 well format. Data can be recorded by luminometeror CCD camera imaging device. The luminescence output is presented asrelative light units (RLU), measured over time.

The anti-proliferative efficacy of Formula I exemplary compounds andcombinations with chemotherapeutic agents are measured by theCellTiter-Glo® Assay (Example 908) against certain hematological tumorcell lines. EC₅₀ values are established for the tested compounds andcombinations.

Exemplary Formula I compounds in Tables 1 and 2 were made,characterized, and tested for inhibition of Btk according to the methodsof this invention, and have the following structures and correspondingnames (ChemDraw Ultra, Version 9.0.1, and ChemBioDraw, Version 11.0,CambridgeSoft Corp., Cambridge Mass.). Where more than one name isassociated with a Formula I compound or intermediate, the chemicalstructure shall define the compound.

TABLE 1 CD69 Hu Blood FACS Mol IC70 No. Structure IUPAC Name Weight (μM)101

(S)-2-(5-fluoro-2- (hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3- yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6- dihydropyridin-3- yl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4- b]indolizin-1(2H)-one 669 0.0338 102

(S)-5-[5-fluoro-2- (hydroxymethyl)-3(1-methyl-5-(5-(2-methyl-4-(oxetan-3- yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6- dihydropyridin-3- yl)phenyl]-8-thia-4,5-diazatricyclo[7.4.0.02,7]trideca- 1(9),2(7),3-trien-6-one 684 0.031 103

(2S)-10-[5-fluoro-2- (hydroxymethyl)-3-[1-methyl-5-({5-[2-methyl-4-(oxetan-3- yl)piperazin-1-yl]pyridine-2-yl}amino)-6-oxo-1,6- dihydropyridin-3-yl]phenyl]- 4,4-dimethyl-1,10-diazatricyclo[6.4.0.02,6]dodeca- 2(6),7-dien-9-one 682 0.0145 104

2-(3-(5-(5-((2S,5R)-2,5- dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2- ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2- (hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydro- pyrazino[1,2-a]indol- 1(2H)-one 682 0.015 105

(S)-2-(3-(5-(5-(2-ethyl-4- (oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl- 6-oxo-1,6-dihydropyridin-3-yl-5-fluoro-2- (hydroxymethyl)phenyl)- 3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol- 1(2H)-one 682 0.008 106

(S)-2-(5-fluoro-2- (hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3- yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6- dihydropyridin-3- yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol- 1(2H)-one 668 0.019 107

(S)-2-(3-(5-(5-(3,4- dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6- dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)- 3,4,6,7,8,9-hexahydro- pyrazino[1,2-a]indol-1(2H)-one 626 0.044 108

(R)-2-(3-(5-(5-(3,4- dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo- 1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl) phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-α]indol- 1(2H)-one 626 0.023 109

(R)-2-(3-(5-(5-(2,4- dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6- dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)- 3,4,6,7,8,9-hexahydro- pyrazino[1,2-a]indol-1(2H)-one 626 0.029 110

(S)-2-(3-(5-(5-(2,4- dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6- dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)- 3,4,6,7,8,9-hexahydro- pyrazino[1,2-a]indol-1(2H)-one 626 0.006 111

2-(5-fluoro-3-(5-(5-(3- (fluoromethyl)-4-methylpiperazin-1-yl)pyridin-2- ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2- (hydroxymethyl)phenyl)- 3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol- 1(2H)-one 644 0.092 112

2-(5-fluoro-2-(hydroxymethyl)- 3-(1-methyl-5-(5-(9-methyl-7-oxa-3,9-diaza- bicyclo[3.3.1]nonan-3- yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)- 3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol- 1(2H)-one 654 0.17 113

(S)-7,7-difluoro-2-(5-fluoro-2- (hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3- yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6- dihydropyridin-3-yl)phenyl)- 3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol- 1(2H)-one 703.75 114

2-(3-{5-[5-((S)-2-Ethyl-4- oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-1-methyl-6- oxo-1,6-dihydro-pyridin-3-yl}-5-fluoro-2-hydroxymethyl- phenyl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H,6H- cyclopenta[4,5]pyrrolo[1,2- a]pyrazin-1-one 695.83 115

(R)-2-(5-fluoro-2- (hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3- yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6- dihydropyridin-3-yl)phenyl)- 3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol- 1(2H)-one 667.77 116

2-(3-{5-[5-((2S,5R)-2,5- Dimethyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2- ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridin-3-yl}-5-fluoro- 2-hydroxymethyl-phenyl)-7,7-dimethyl-3,4,7,8-tetrahydro- 2H,6H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1-one 695.83 117

3-(5-Fluoro-2-hydroxymethyl-3- {1-methyl-5-[5-((R)-2-methyl-4-oxetan-3-yl-piperazin-1-yl)- pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)- 6,7,8,9-tetrahydro-3H-benzo[4,5]thieno[2,3-d] pyridazin-4-one 683.79 118

3-(3-{5-[5-((2S,5R)-2,5- Dimethyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2- ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridin-3-yl}-5-fluoro- 2-hydroxymethyl-phenyl)-6,7,8,9-tetrahydro-3H- benzo[4,5]thieno[2,3-d] pyridazin-4-one 697.82119

(S)-10-fluoro-2-(5-fluoro-2- (hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3- yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydro- pyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol- 1(2H)-one 685.76 120

3-(3-{5-[5-((S)-2-Ethyl-4 oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-1-methyl-6- oxo-1,6-dihydro-pyridin-3-yl}-5-fluoro-2-hydroxymethyl- phenyl)-6,7,8,9-tetrahydro-3H-benzo[4,5]thieno[2,3- d]pyridazin-4-one 697.82 121

2-(3-(5-(5-((2S,5R)-2,5- dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2- ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2- (hydroxymethyl)phenyl)-6,7,8,9-tetrahydropyrazino[1,2-a]indol- 1(2H)-one 679.78 122

(S)-2-(3-(5-(5-(2-ethyl-4- (oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl- 6-oxo-1,6-dihyropyridin-3-yl)-5-fluoro-2-(hydroxy- methyl)phenyl)-6,7,8,9-tetrahydropyrazino[1,2-a]indol- 1(2H)-one 680.78 123

(S)-2-(3-(5-(5-(2-ethyl-4- (oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl- 6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxy- methyl)phenyl)-3,4,6,7,8,9- hexahydropyrido[3,4-b]indolizin-1(2H)-one 681.80 124

2-(5-Fluoro-2-hydroxymethyl-3- {1-methyl-5-[5-((R)-2-methyl-4-oxetan-3-yl-piperazin-1-yl)- pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)- 7,7-dimethyl-3,4,7,8-tetrahydro-2H,6H-cyclopenta[4,5] pyrrolo[1,2-a]pyrazin-1-one 681.80 125

2-(3-(5-(5-(2,2-dimethyl-4- (oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl- 6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxy- methyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol- 1(2H)-one 681.80

TABLE 2 CD69 Hu Blood FACS No. Structure IUPAC Name (IC70) 126

2-[3-[5-[[5-[(2S,5R)-2,5- dimethyl-4-(oxetan-3- yl)piperazin-1-yl]-2-pyridyl]amino]-1-methyl-6-oxo- 3-pyridyl]-5-fluoro-2-(hydroxymethyl)phenyl]- 3,4,6,7,8,9- hexahydropyrido[3,4-b]indolizin-1-one 0.0571 127

2-[5-fluoro-2-(hydroxymethyl)- 3-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin- 1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]phenyl]-6,7,8,9- tetrahydropyridazino[4,5- b]indolizin-1-one0.051 128

2-(7,7-dimethyl-4-oxo-1,2,6,8- tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-4-fluoro-6- [1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2- pyridyl]amino]-6-oxo-3-pyridyl]benzoic acid 5 129

2-(7,7-dimethyl-4-oxo-1,2,6,8- tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-4-fluoro-N- methyl-6-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3- yl)piperazin-1-yl]-2- pyridyl]amino]-6-oxo-3-pyridyl]benzamide 0.472 130

3-[2-(hydroxymethyl)-3-[1- methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2- pyridyl]amino]-6-oxo-3-pyridyl]phenyl]-6,7,8,9- tetrahydrobenzothiopheno[2,3- d]pyridazin-4-one0.0354 131

3-[5-fluoro-2-(hydroxymethyl)- 3-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin- 1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]phenyl]-7,7-dimethyl- 6,8-dihydrocyclopenta[3,4]thieno[1,3-d]pyridazin-4-one 0.0349Administration of Formula I Compounds

The compounds of the invention may be administered by any routeappropriate to the condition to be treated. Suitable routes includeoral, parenteral (including subcutaneous, intramuscular, intravenous,intraarterial, intradermal, intrathecal and epidural), transdermal,rectal, nasal, topical (including buccal and sublingual), vaginal,intraperitoneal, intrapulmonary and intranasal. For localimmunosuppressive treatment, the compounds may be administered byintralesional administration, including perfusing or otherwisecontacting the graft with the inhibitor before transplantation. It willbe appreciated that the preferred route may vary with for example thecondition of the recipient. Where the compound is administered orally,it may be formulated as a pill, capsule, tablet, etc. with apharmaceutically acceptable carrier or excipient. Where the compound isadministered parenterally, it may be formulated with a pharmaceuticallyacceptable parenteral vehicle and in a unit dosage injectable form, asdetailed below.

A dose to treat human patients may range from about 10 mg to about 1000mg of Formula I compound. A typical dose may be about 100 mg to about300 mg of the compound. A dose may be administered once a day (QID),twice per day (BID), or more frequently, depending on thepharmacokinetic and pharmacodynamic properties, including absorption,distribution, metabolism, and excretion of the particular compound. Inaddition, toxicity factors may influence the dosage and administrationregimen. When administered orally, the pill, capsule, or tablet may beingested daily or less frequently for a specified period of time. Theregimen may be repeated for a number of cycles of therapy.

Methods of Treatment with Formula I Compounds

Formula I compounds of the present invention are useful for treating ahuman or animal patient suffering from a disease or disorder arisingfrom abnormal cell growth, function or behavior associated with Btkkinase such as an immune disorder, cardiovascular disease, viralinfection, inflammation, a metabolism/endocrine disorder or aneurological disorder, may thus be treated by a method comprising theadministration thereto of a compound of the present invention as definedabove. A human or animal patient suffering from cancer may also betreated by a method comprising the administration thereto of a compoundof the present invention as defined above. The condition of the patientmay thereby be improved or ameliorated.

Formula I compounds may be useful for in vitro, in situ, and in vivodiagnosis or treatment of mammalian cells, organisms, or associatedpathological conditions, such as systemic and local inflammation,immune-inflammatory diseases such as rheumatoid arthritis, immunesuppression, organ transplant rejection, allergies, ulcerative colitis,Crohn's disease, dermatitis, asthma, systemic lupus erythematosus,Sjögren's Syndrome, multiple sclerosis, scleroderma/systemic sclerosis,idiopathic thrombocytopenic purpura (ITP), anti-neutrophil cytoplasmicantibodies (ANCA) vasculitis, chronic obstructive pulmonary disease(COPD), psoriasis, and for general joint protective effects.

Methods of the invention also include treating such diseases asarthritic diseases, such as rheumatoid arthritis, monoarticulararthritis, osteoarthritis, gouty arthritis, spondylitis; Behcet disease;sepsis, septic shock, endotoxic shock, gram negative sepsis, grampositive sepsis, and toxic shock syndrome; multiple organ injurysyndrome secondary to septicemia, trauma, or hemorrhage; ophthalmicdisorders such as allergic conjunctivitis, vernal conjunctivitis,uveitis, and thyroid-associated ophthalmopathy; eosinophilic granuloma;pulmonary or respiratory disorders such as asthma, chronic bronchitis,allergic rhinitis, ARDS, chronic pulmonary inflammatory disease (e.g.,chronic obstructive pulmonary disease), silicosis, pulmonarysarcoidosis, pleurisy, alveolitis, vasculitis, emphysema, pneumonia,bronchiectasis, and pulmonary oxygen toxicity; reperfusion injury of themyocardium, brain, or extremities; fibrosis such as cystic fibrosis;keloid formation or scar tissue formation; atherosclerosis; autoimmunediseases, such as systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, some forms of diabetes, and Reynaud'ssyndrome; and transplant rejection disorders such as GVHD and allograftrejection; chronic glomerulonephritis; inflammatory bowel diseases suchas chronic inflammatory bowel disease (CIBD), Crohn's disease,ulcerative colitis, and necrotizing enterocolitis; inflammatorydermatoses such as contact dermatitis, atopic dermatitis, psoriasis, orurticaria; fever and myalgias due to infection; central or peripheralnervous system inflammatory disorders such as meningitis, encephalitis,and brain or spinal cord injury due to minor trauma; Sjogren's syndrome;diseases involving leukocyte diapedesis; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases; hypovolemicshock; Type I diabetes mellitus; acute and delayed hypersensitivity;disease states due to leukocyte dyscrasia and metastasis; thermalinjury; granulocyte transfusion-associated syndromes; andcytokine-induced toxicity.

Methods of the invention also include treating cancer selected frombreast, ovary, cervix, prostate, testis, genitourinary tract, esophagus,larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma,lung, epidermoid carcinoma, large cell carcinoma, non-small cell lungcarcinoma (NSCLC), small cell carcinoma, lung adenocarcinoma, bone,colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma,undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma,sarcoma, bladder carcinoma, liver carcinoma and biliary passages, kidneycarcinoma, pancreatic, myeloid disorders, lymphoma, hairy cells, buccalcavity, naso-pharyngeal, pharynx, lip, tongue, mouth, small intestine,colon-rectum, large intestine, rectum, brain and central nervous system,Hodgkin's, leukemia, bronchus, thyroid, liver and intrahepatic bileduct, hepatocellular, gastric, glioma/glioblastoma, endometrial,melanoma, kidney and renal pelvis, urinary bladder, uterine corpus,uterine cervix, multiple myeloma, acute myelogenous leukemia, chronicmyelogenous leukemia, lymphocytic leukemia, chronic lymphoid leukemia(CLL), myeloid leukemia, oral cavity and pharynx, non-Hodgkin lymphoma,melanoma, and villous colon adenoma.

The methods of the invention can have utility in treating subjects whoare or can be subject to reperfusion injury, i.e., injury resulting fromsituations in which a tissue or organ experiences a period of ischemiafollowed by reperfusion. The term “ischemia” refers to localized tissueanemia due to obstruction of the inflow of arterial blood. Transientischemia followed by reperfusion characteristically results inneutrophil activation and transmigration through the endothelium of theblood vessels in the affected area. Accumulation of activatedneutrophils in turn results in generation of reactive oxygenmetabolites, which damage components of the involved tissue or organ.This phenomenon of “reperfusion injury” is commonly associated withconditions such as vascular stroke (including global and focalischemia), hemorrhagic shock, myocardial ischemia or infarction, organtransplantation, and cerebral vasospasm. To illustrate, reperfusioninjury occurs at the termination of cardiac bypass procedures or duringcardiac arrest when the heart, once prevented from receiving blood,begins to reperfuse. It is expected that inhibition of Btk activity mayresult in reduced amounts of reperfusion injury in such situations.

Pharmaceutical Formulations

In order to use a compound of this invention for the therapeutictreatment of mammals including humans, it is normally formulated inaccordance with standard pharmaceutical practice as a pharmaceuticalcomposition. According to this aspect of the invention there is provideda pharmaceutical composition comprising a compound of this invention inassociation with a pharmaceutically acceptable diluent or carrier.

A typical formulation is prepared by mixing a compound of the presentinvention and a carrier, diluent or excipient. Suitable carriers,diluents and excipients are well known to those skilled in the art andinclude materials such as carbohydrates, waxes, water soluble and/orswellable polymers, hydrophilic or hydrophobic materials, gelatin, oils,solvents, water and the like. The particular carrier, diluent orexcipient used will depend upon the means and purpose for which thecompound of the present invention is being applied. Solvents aregenerally selected based on solvents recognized by persons skilled inthe art as safe (GRAS) to be administered to a mammal. In general, safesolvents are non-toxic aqueous solvents such as water and othernon-toxic solvents that are soluble or miscible in water. Suitableaqueous solvents include water, ethanol, propylene glycol, polyethyleneglycols (e.g., PEG 400, PEG 300), etc. and mixtures thereof. Theformulations may also include one or more buffers, stabilizing agents,surfactants, wetting agents, lubricating agents, emulsifiers, suspendingagents, preservatives, antioxidants, opaquing agents, glidants,processing aids, colorants, sweeteners, perfuming agents, flavoringagents and other known additives to provide an elegant presentation ofthe drug (i.e., a compound of the present invention or pharmaceuticalcomposition thereof) or aid in the manufacturing of the pharmaceuticalproduct (i.e., medicament).

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described above. The compound of the present inventionis typically formulated into pharmaceutical dosage forms to provide aneasily controllable dosage of the drug and to enable patient compliancewith the prescribed regimen.

The pharmaceutical composition (or formulation) for application may bepackaged in a variety of ways depending upon the method used foradministering the drug. Generally, an article for distribution includesa container having deposited therein the pharmaceutical formulation inan appropriate form. Suitable containers are well known to those skilledin the art and include materials such as bottles (plastic and glass),sachets, ampoules, plastic bags, metal cylinders, and the like. Thecontainer may also include a tamper-proof assemblage to preventindiscreet access to the contents of the package. In addition, thecontainer has deposited thereon a label that describes the contents ofthe container. The label may also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration. Forexample, a compound of Formula I having the desired degree of purity mayoptionally be mixed with pharmaceutically acceptable diluents, carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences (1980)16th edition, Osol, A. Ed.), in the form of a lyophilized formulation,milled powder, or an aqueous solution. Formulation may be conducted bymixing at ambient temperature at the appropriate pH, and at the desireddegree of purity, with physiologically acceptable carriers, i.e.,carriers that are non-toxic to recipients at the dosages andconcentrations employed. The pH of the formulation depends mainly on theparticular use and the concentration of compound, but may range fromabout 3 to about 8. Formulation in an acetate buffer at pH 5 is asuitable embodiment.

The compound ordinarily can be stored as a solid composition, alyophilized formulation or as an aqueous solution.

The pharmaceutical compositions of the invention will be formulated,dosed and administered in a fashion, i.e., amounts, concentrations,schedules, course, vehicles and route of administration, consistent withgood medical practice. Factors for consideration in this context includethe particular disorder being treated, the particular mammal beingtreated, the clinical condition of the individual patient, the cause ofthe disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the compound to be administered will be governed by suchconsiderations, and is the minimum amount necessary to ameliorate, ortreat the hyperproliferative disorder.

As a general proposition, the initial pharmaceutically effective amountof the inhibitor administered parenterally per dose will be in the rangeof about 0.01-100 mg/kg, namely about 0.1 to 20 mg/kg of patient bodyweight per day, with the typical initial range of compound used being0.3 to 15 mg/kg/day.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theactive pharmaceutical ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations of compounds of Formula I may beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing acompound of Formula I, which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate) and poly-D-(−)-3-hydroxybutyric acid.

The formulations include those suitable for the administration routesdetailed herein. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Techniques and formulations generally are found inRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).Such methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations of a compound of Formula I suitable for oral administrationmay be prepared as discrete units such as pills, capsules, cachets ortablets each containing a predetermined amount of a compound of FormulaI. Compressed tablets may be prepared by compressing in a suitablemachine the active ingredient in a free-flowing form such as a powder orgranules, optionally mixed with a binder, lubricant, inert diluent,preservative, surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and optionally are formulated so as toprovide slow or controlled release of the active ingredient therefrom.Tablets, troches, lozenges, aqueous or oil suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, e.g., gelatincapsules, syrups or elixirs may be prepared for oral use. Formulationsof compounds of Formula I intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents including sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide a palatablepreparation. Tablets containing the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipient which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium or sodium carbonate, lactose,calcium or sodium phosphate; granulating and disintegrating agents, suchas maize starch, or alginic acid; binding agents, such as starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc. Tablets may be uncoated or may be coated by knowntechniques including microencapsulation to delay disintegration andadsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

For treatment of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w. When formulated in an ointment, the active ingredientsmay be employed with either a paraffinic or a water-miscible ointmentbase. Alternatively, the active ingredients may be formulated in a creamwith an oil-in-water cream base. If desired, the aqueous phase of thecream base may include a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane 1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400)and mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the activeingredient through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethyl sulfoxide and relatedanalogs. The oily phase of the emulsions of this invention may beconstituted from known ingredients in a known manner. While the phasemay comprise merely an emulsifier, it desirably comprises a mixture ofat least one emulsifier with a fat or an oil or with both a fat and anoil. Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make up the so-called emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations.Emulsifiers and emulsion stabilizers suitable for use in the formulationof the invention include Tween® 60, Span® 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate.

Aqueous suspensions of Formula I compounds contain the active materialsin admixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, croscarmellose, povidone, methylcellulose,hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone,gum tragacanth and gum acacia, and dispersing or wetting agents such asa naturally occurring phosphatide (e.g., lecithin), a condensationproduct of an alkylene oxide with a fatty acid (e.g., polyoxyethylenestearate), a condensation product of ethylene oxide with a long chainaliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such as sucroseor saccharin.

The pharmaceutical compositions of compounds of Formula I may be in theform of a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents which have been mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butanediol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to 20% w/w, for exampleabout 0.5 to 10% w/w, for example about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis disorders as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

Combination Therapy

The compounds of Formula I may be employed alone or in combination withother therapeutic agents for the treatment of a disease or disorderdescribed herein, such as inflammation or a hyperproliferative disorder(e.g., cancer). In certain embodiments, a compound of Formula I iscombined in a pharmaceutical combination formulation, or dosing regimenas combination therapy, with a second therapeutic compound that hasanti-inflammatory or anti-hyperproliferative properties or that isuseful for treating an inflammation, immune-response disorder, orhyperproliferative disorder (e.g., cancer). The second therapeutic agentmay be an NSAID anti-inflammatory agent. The second therapeutic agentmay be a chemotherapeutic agent. The second compound of thepharmaceutical combination formulation or dosing regimen preferably hascomplementary activities to the compound of Formula I such that they donot adversely affect each other. Such compounds are suitably present incombination in amounts that are effective for the purpose intended. Inone embodiment, a composition of this invention comprises a compound ofFormula I, or a stereoisomer, tautomer, solvate, metabolite, orpharmaceutically acceptable salt or prodrug thereof, in combination witha therapeutic agent such as an NSAID.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other therapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e., the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g., by different injections in separate syringes,separate pills or capsules, or separate infusions. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially, whereas in combinationtherapy, effective dosages of two or more active ingredients areadministered together.

In a particular embodiment of therapy, a compound of Formula I, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof, may be combined with othertherapeutic, hormonal or antibody agents such as those described herein,as well as combined with surgical therapy and radiotherapy. Combinationtherapies according to the present invention thus comprise theadministration of at least one compound of Formula I, or a stereoisomer,tautomer, solvate, metabolite, or pharmaceutically acceptable salt orprodrug thereof, and the use of at least one other cancer treatmentmethod. The amounts of the compound(s) of Formula I and the otherpharmaceutically active therapeutic agent(s) and the relative timings ofadministration will be selected in order to achieve the desired combinedtherapeutic effect.

Metabolites of Compounds of Formula I

Also falling within the scope of this invention are the in vivometabolic products of Formula I described herein. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,deamidation, esterification, deesterification, enzymatic cleavage, andthe like, of the administered compound. Accordingly, the inventionincludes metabolites of compounds of Formula I, including compoundsproduced by a process comprising contacting a compound of this inventionwith a mammal for a period of time sufficient to yield a metabolicproduct thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g., ¹⁴C or ³H) isotope of a compound of the invention,administering it parenterally in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours) and isolating its conversion products fromthe urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS, LC/MS or NMR analysis. In general, analysis of metabolites is donein the same way as conventional drug metabolism studies well known tothose skilled in the art. The metabolite products, so long as they arenot otherwise found in vivo, are useful in diagnostic assays fortherapeutic dosing of the compounds of the invention.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the diseases anddisorders described above is provided. In one embodiment, the kitcomprises a container comprising a compound of Formula I, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof. The kit may further comprise a labelor package insert on or associated with the container. The term “packageinsert” is used to refer to instructions customarily included incommercial packages of therapeutic products, that contain informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such therapeutic products.Suitable containers include, for example, bottles, vials, syringes,blister pack, etc. The container may be formed from a variety ofmaterials such as glass or plastic. The container may hold a compound ofFormula I or a formulation thereof which is effective for treating thecondition and may have a sterile access port (for example, the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a compound of Formula I. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. In addition, the label or package insert mayindicate that the patient to be treated is one having a disorder such asa hyperproliferative disorder, neurodegeneration, cardiac hypertrophy,pain, migraine or a neurotraumatic disease or event. In one embodiment,the label or package inserts indicates that the composition comprising acompound of Formula I can be used to treat a disorder resulting fromabnormal cell growth. The label or package insert may also indicate thatthe composition can be used to treat other disorders. Alternatively, oradditionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The kit may further comprise directions for the administration of thecompound of Formula I and, if present, the second pharmaceuticalformulation. For example, if the kit comprises a first compositioncomprising a compound of Formula I and a second pharmaceuticalformulation, the kit may further comprise directions for thesimultaneous, sequential or separate administration of the first andsecond pharmaceutical compositions to a patient in need thereof.

In another embodiment, the kits are suitable for the delivery of solidoral forms of a compound of Formula I, such as tablets or capsules. Sucha kit preferably includes a number of unit dosages. Such kits caninclude a card having the dosages oriented in the order of theirintended use. An example of such a kit is a “blister pack”. Blisterpacks are well known in the packaging industry and are widely used forpackaging pharmaceutical unit dosage forms. If desired, a memory aid canbe provided, for example in the form of numbers, letters, or othermarkings or with a calendar insert, designating the days in thetreatment schedule in which the dosages can be administered.

According to one embodiment, a kit may comprise (a) a first containerwith a compound of Formula I contained therein; and optionally (b) asecond container with a second pharmaceutical formulation containedtherein, wherein the second pharmaceutical formulation comprises asecond compound with anti-hyperproliferative activity. Alternatively, oradditionally, the kit may further comprise a third container comprisinga pharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

In certain other embodiments wherein the kit comprises a composition ofFormula I and a second therapeutic agent, the kit may comprise acontainer for containing the separate compositions such as a dividedbottle or a divided foil packet, however, the separate compositions mayalso be contained within a single, undivided container. Typically, thekit comprises directions for the administration of the separatecomponents. The kit form is particularly advantageous when the separatecomponents are preferably administered in different dosage forms (e.g.,oral and parenteral), are administered at different dosage intervals, orwhen titration of the individual components of the combination isdesired by the prescribing physician.

Preparation of Formula I Compounds

Compounds of Formula I may be synthesized by synthetic routes thatinclude processes analogous to those well-known in the chemical arts,particularly in light of the description contained herein, and those forother heterocycles described in: Comprehensive Heterocyclic ChemistryII, Editors Katritzky and Rees, Elsevier, 1997, e.g. Volume 3; LiebigsAnnalen der Chemie, (9):1910-16, (1985); Helvetica Chimica Acta,41:1052-60, (1958); Arzneimittel-Forschung, 40(12):1328-31, (1990), eachof which are expressly incorporated by reference. Starting materials aregenerally available from commercial sources such as Aldrich Chemicals(Milwaukee, Wis.) or are readily prepared using methods well known tothose skilled in the art (e.g., prepared by methods generally describedin Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v.1-23, Wiley, N.Y. (1967-2006 ed.), or Beilsteins Handbuch derorganischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, includingsupplements (also available via the Beilstein online database).

Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing Formula I compoundsand necessary reagents and intermediates are known in the art andinclude, for example, those described in R. Larock, ComprehensiveOrganic Transformations, VCH Publishers (1989); T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., John Wileyand Sons (1999); and L. Paquette, ed., Encyclopedia of Reagents forOrganic Synthesis, John Wiley and Sons (1995) and subsequent editionsthereof.

Compounds of Formula I may be prepared singly or as compound librariescomprising at least 2, for example 5 to 1,000 compounds, or 10 to 100compounds. Libraries of compounds of Formula I may be prepared by acombinatorial ‘split and mix’ approach or by multiple parallel synthesesusing either solution phase or solid phase chemistry, by proceduresknown to those skilled in the art. Thus according to a further aspect ofthe invention there is provided a compound library comprising at least 2compounds, or pharmaceutically acceptable salts thereof.

The Figures and Examples provide exemplary methods for preparing FormulaI compounds. Those skilled in the art will appreciate that othersynthetic routes may be used to synthesize the Formula I compounds.Although specific starting materials and reagents are depicted anddiscussed in the Figures and Examples, other starting materials andreagents can be easily substituted to provide a variety of derivativesand/or reaction conditions. In addition, many of the exemplary compoundsprepared by the described methods can be further modified in light ofthis disclosure using conventional chemistry well known to those skilledin the art.

In preparing compounds of Formulas I, protection of remote functionality(e.g., primary or secondary amine) of intermediates may be necessary.The need for such protection will vary depending on the nature of theremote functionality and the conditions of the preparation methods.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such protection isreadily determined by one skilled in the art. For a general descriptionof protecting groups and their use, see T. W. Greene, Protective Groupsin Organic Synthesis, John Wiley & Sons, New York, 1991.

Experimental procedures, intermediates and reagents useful for usefulfor the preparation of Formula I compounds may be found in U.S. Ser. No.13/102,720, “PYRIDONE AND AZA-PYRIDONE COMPOUNDS AND METHODS OF USE”,filed 6 May 2011, which is incorporated by reference in its entirety.

FIGS. 1-12 describe the synthesis of exemplary embodiments of Formula Icompounds 101-125, more fully described in Examples 101-112, and may beuseful for the preparation of other Formula I compounds.

GENERAL PREPARATIVE PROCEDURES General Procedure: Suzuki Coupling

The Suzuki-type coupling reaction is useful to form carbon-carbon bondsto attach the rings of Formula I compounds and intermediates such as A-3(Suzuki (1991) Pure Appl. Chem. 63:419-422; Miyaura and Suzuki (1979)Chem. Reviews 95(7):2457-2483; Suzuki (1999) J. Organometal. Chem.576:147-168). Suzuki coupling is a palladium mediated cross couplingreaction of an arylhalide, such as B-2 or B-4, with a boronic acid suchas A-1 or A-2. For example, B-2 may be combined with about 1.5equivalents of4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane), anddissolved in about 3 equivalents of sodium carbonate as a 1 molarsolution in water and an equal volume of acetonitrile. A catalyticamount, or more, of a low valent palladium reagent, such asbis(triphenylphosphine)palladium(II) dichloride, is added. In some casespotassium acetate is used in place of sodium carbonate to adjust the pHof the aqueous layer. The reaction is then heated to about 140-150° C.under pressure in a microwave reactor (Biotage AB, Uppsala, Sweden) for10 to 30 minutes. The contents are extracted with ethyl acetate, oranother organic solvent. After evaporation of the organic layer theboron ester A-1 may be purified on silica or by reverse phase HPLC.Substituents are as defined, or protected forms or precursors thereof.Likewise, bromide intermediate B-4 can be boronylated to give A-2.Suzuki coupling of B-2 and A-2, or of A-1 and B-4, gives Formula Icompound or intermediate A-3. Boronic ester (or acid) (1.5 eq) A-1 orA-2, and a palladium catalyst such asbis(triphenylphosphine)palladium(II) chloride (0.05 eq) is added to amixture of halo intermediate (1 eq) B-2 or B-4 in acetonitrile and 1 Mof sodium carbonate aqueous solution (equal volume as acetonitrile). Thereaction mixture is heated to about 150° C. in a microwave for about 15min. LC/MS indicates when the reaction is complete. Water is added tothe mixture, and the precipitated product is filtered and purified byHPLC to yield the product A-3. Substituents are as defined, or protectedforms or precursors thereof.

A variety of palladium catalysts can be used during the Suzuki couplingstep. Various low valent, Pd(II) and Pd(0) catalysts may be used in theSuzuki coupling reaction, including PdCl2(PPh₃)₂, Pd(t-Bu)₃, PdCl₂ dppfCH₂Cl₂, Pd(PPh₃)₄, Pd(OAc)/PPh₃, Cl₂Pd[(Pet₃)]₂, Pd(DIPHOS)₂,Cl₂Pd(Bipy), [PdCl(Ph₂PCH₂PPh₂)]₂, Cl₂Pd[P(o-tol)₃]₂,Pd₂(dba)₃/P(o-tol)₃, Pd₂(dba)/P(furyl)₃, Cl₂Pd[P(furyl)₃]₂,Cl₂Pd(PMePh₂)₂, Cl₂Pd[P(4-F-Ph)₃]₂, Cl₂Pd[P(C₆F₆)₃]₂,Cl₂Pd[P(2-COOH-Ph)(Ph)₂]₂, Cl₂Pd[P(4-COOH-Ph)(Ph)₂]₂, and encapsulatedcatalysts Pd EnCat™ 30, Pd EnCat™ TPP30, and Pd(II)EnCat™ BINAP30 (US2004/0254066).

General Procedure: Buchwald reaction

The Buchwald reaction is useful to aminate 6-bromo intermediates B-1(Wolf and Buchwald (2004) Org. Synth Coll. Vol. 10:423; Paul et al(1994) Jour. Amer. Chem. Soc. 116:5969-5970). To a solution of3,5-dihalo-pyridone intermediate B-1 in DMF is added the appropriate5-(piperazin-1-yl)pyridin-2-amine, e.g. 104b, or5-(piperazin-1-yl)pyrazin-2-amine (200 mol %), Cs₂CO₃ (50 mol %),Pd₂(dba)₃ (5 mol %), and 4,5-bis(diphenylphosphino)-9,9-dimethyxanthene(Xantphos, CAS Reg. No. 161265-03-8, 10 mol %). The reaction is heatedto about 110° C. under pressure in a microwave reactor (Biotage AB,Uppsala, Sweden) for about 30 min. The resulting solution isconcentrated in vacuo to give B-2. Other palladium catalysts andphosphine ligands may be useful.

N-Aryl amide intermediates B-4 can also be prepared under Buchwaldconditions with cyclic amide intermediates (R⁷) such as3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 101e and arylbromides B-3.

Methods of Separation

In the methods of preparing Formula I compounds, it may be advantageousto separate reaction products from one another and/or from startingmaterials. The desired products of each step or series of steps isseparated and/or purified to the desired degree of homogeneity by thetechniques common in the art. Typically such separations involvemultiphase extraction, crystallization from a solvent or solventmixture, distillation, sublimation, or chromatography. Chromatographycan involve any number of methods including, for example: reverse-phaseand normal phase; size exclusion; ion exchange; high, medium and lowpressure liquid chromatography methods and apparatus; small scaleanalytical; simulated moving bed (SMB) and preparative thin or thicklayer chromatography, as well as techniques of small scale thin layerand flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like. Selection of appropriate methodsof separation depends on the nature of the materials involved, such as,boiling point and molecular weight in distillation and sublimation,presence or absence of polar functional groups in chromatography,stability of materials in acidic and basic media in multiphaseextraction, and the like.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present invention may be atropisomers(e.g., substituted biaryls) and are considered as part of thisinvention. Enantiomers can also be separated by use of a chiral HPLCcolumn.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S. “Stereochemistry of OrganicCompounds,” John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,(1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of chiralcompounds of the invention can be separated and isolated by any suitablemethod, including: (1) formation of ionic, diastereomeric salts withchiral compounds and separation by fractional crystallization or othermethods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: “DrugStereochemistry, Analytical Methods and Pharmacology,” Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-phenylethylamine (amphetamine), and the like withasymmetric compounds bearing acidic functionality, such as carboxylicacid and sulfonic acid. The diastereomeric salts may be induced toseparate by fractional crystallization or ionic chromatography. Forseparation of the optical isomers of amino compounds, addition of chiralcarboxylic or sulfonic acids, such as camphorsulfonic acid, tartaricacid, mandelic acid, or lactic acid can result in formation of thediastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(E. and Wilen, S. “Stereochemistry of Organic Compounds”, John Wiley &Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed byreacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the pure orenriched enantiomer. A method of determining optical purity involvesmaking chiral esters, such as a menthyl ester, e.g., (−) menthylchloroformate in the presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem.(1982) 47:4165), of the racemic mixture, and analyzing the ¹H NMRspectrum for the presence of the two atropisomeric enantiomers ordiastereomers. Stable diastereomers of atropisomeric compounds can beseparated and isolated by normal- and reverse-phase chromatographyfollowing methods for separation of atropisomeric naphthyl-isoquinolines(WO 96/15111). By method (3), a racemic mixture of two enantiomers canbe separated by chromatography using a chiral stationary phase (“ChiralLiquid Chromatography” (1989) W. J. Lough, Ed., Chapman and Hall, NewYork; Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purifiedenantiomers can be distinguished by methods used to distinguish otherchiral molecules with asymmetric carbon atoms, such as optical rotationand circular dichroism.

EXAMPLES Example 101a 2,6-Dibromo-4-fluorobenzaldehyde 101a

A solution of 1,3-dibromo-5-fluoro-2-iodobenzene (50 g, 132 mmol) inanhydrous toluene (300 mL) was cooled to −35° C. A solution ofisopropylmagnesium chloride (84 mL, 171 mmol, 2.0 M in diethyl ether)over a period of 30 minutes while maintaining the internal temperaturebelow −25° C. See FIG. 1. A clear brown solution was obtained. Stirringwas continued for 1.5 h. Then anhydrous DMF (34 mL, 436 mmol) was addedover a period of 30 minutes. The temperature of the reaction mixtureincreased to −19° C. The reaction mixture was warmed to 10° C. (roomtemperature) over 1 h and stirred at this temperature for 1.5 h. Thereaction was quenched with saturated aqueous NH₄Cl (100 mL), filteredand evaporated under reduced pressure. The residue was purified bysilica-gel column chromatography (eluting with petroleum ether/ethylacetate:from 50:1 to 20:1) to give 101a (20 g, yield 54%) as a yellowsolid.

Example 101b 2,6-Dibromo-4-fluorophenyl)methanol 101b

A solution of 2,6-dibromo-4-fluorobenzaldehyde 101a (20 g, 71 mmol) inethanol (500 mL) was added NaBH₄ (10 g, 284 mmol). The mixture wasstirred at room temperature (10° C.) for 4 h. TLC showed the startmaterial disappeared. The reaction was qunched with HCl solution (150mL, 1 M). Most of ethanol was evaporated at reduced pressure. Theresidue was extracted by ethyl acetate (3×500 mL). The organic layerswas combined and dried with anhy. Na₂SO₄, evaporated in vacuo. Theresidue was purified by silica-gel column chromatography (eluting withpetroleum ether/ethyl acetate:from 50:1 to 20:1) to give 101b (15 g,yield 75%) as a white solid.

Example 101c 2,6-Dibromo-4-fluorobenzyl acetate 101c

A 500-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was charged with 101b (23.0 g, 81.0 mmol),triethyl amine (25.0 g, 247 mmol) in anhydrous methylene chloride (100mL). Acetic anhydride (10.0 g, 98.0 mmol) was added and this mixture wasstirred at room temperature for 16 h. After this time, the mixture wasdiluted with methylene chloride (100 mL) and washed with saturatedaqueous sodium bicarbonate (100 mL). The layers were separated and theaqueous layer was extracted with methylene chloride (2×20 mL). Theorganic extracts were combined and dried over sodium sulfate. The dryingagent was removed by filtration. The filtrate was concentrated underreduced pressure, and the resulting residue was purified by flash columnchromatography (silica, 0% to 50% ethyl acetate/hexanes) to afford 101cin 87% yield (23.0 g) as a white solid.

Example 101d Methyl 5,6,7,8-Tetrahydroindolizine-2-carboxylate 112d

A 500-mL round-bottomed flask equipped with a magnetic stirrer andnitrogen inlet was purged with nitrogen and charged with5,6,7,8-tetrahydroindolizine-2-carboxylic acid (30.4 g, 184 mmol), DMF(1.00 g, 13.6 mmol) and methylene chloride (300 mL). The solution wascooled to 0° C. using an ice bath. Oxalyl chloride (28.0 g, 221 mmol)was added dropwise, and the reaction mixture was warmed to roomtemperature over 30 min and stirred for 5 h. After this time, theresulting solution was concentrated to afford a brown solid. This solidwas dissolved in anhydrous methanol (400 mL), and the solution wascooled to 0° C. Triethylamine (57 g, 552 mmol) was added to the reactionmixture, and it was stirred for a further 2 h at room temperature. Afterthis time, the reaction mixture was concentrated to dryness underreduced pressure. The residue was diluted with methylene chloride (300mL) and washed with water (200 mL) and saturated aqueous sodiumbicarbonate (200 mL). The organic layer was dried over sodium sulfate,filtered and concentrated under reduced pressure. The resulting residuewas titrated with hexane (200 mL) to afford 101d in 58% yield (19.1 g)as a white solid: mp 72-74° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 7.13 (s,1H), 6.23 (s, 1H), 3.93 (t, 2H, J=6.0 Hz), 3.77 (s, 3H), 2.75 (t, 2H,J=6.0 Hz), 1.93 (m, 2H), 1.80 (m, 2H); (APCI+) m/z 180.1 (M+H)

Example 101e Methyl3-(Cyanomethyl)-5,6,7,8-tetrahydroindolizine-2-carboxylate 101e

A 500-mL three-neck round-bottomed flask equipped with an additionfunnel, thermometer and charged with methyl5,6,7,8-tetrahydroindolizine-2-carboxylate 101d (6.70 g, 37.4 mmol),iodoacetonitrile (12.5 g, 74.9 mmol), iron (II) sulfate heptahydrate(5.20 g, 18.7 mmol) and dimethyl sulfoxide (250 mL). Hydrogen peroxide(35%, 18.2 g, 187 mmol) was added dropwise to the mixture in 1 h througha syringe pump at room temperature using a water bath. Iron (II) sulfateheptahydrate (2 to 3 equivalent) was added to the reaction mixture inportions to keep the temperature between 25° C. to 35° C., until thecolor of the reaction mixture is deep red. If TLCs show the reaction notcompleted, then more hydrogen peroxide (2-3 equivalent) and more iron(II) sulfate heptahydrate (1-2 equivalent) were added in the same manneruntil the reaction is completed. After that time, the reaction mixturewas partitioned between aqueous saturated sodium bicarbonate solution(200 mL) and ethyl acetate (400 mL). The organic layer was separated,and the aqueous layer was extracted with ethyl acetate (2×100 mL). Thecombined organic layers were washed with saturated Sodium thiosulfatesolution (50 mL), dried over sodium sulfate and concentrated underreduced pressure. The residue was purified by column chromatography toafford a 78% yield (6.40 g) of 101e as a yellow oil: ¹H NMR (500 MHz,CDCl₃) δ 6.23 (s, 1H), 4.23 (s, 2H), 3.94 (t, 2H, J=6.5 Hz), 3.81 (s,3H), 2.74 (t, 2H, J=6.5 Hz), 2.00 (m, 2H), 1.83 (m, 2H); (APCI+) m/z219.3 (M+H)

Example 101f Methyl3-(2-Aminoethyl)-5,6,7,8-tetrahydroindolizine-2-carboxylate HydrogenChloride Salt 101f

Methyl 3-(Cyanomethyl)-5,6,7,8-tetrahydroindolizine-2-carboxylate 101ewas hydrogenated with platinum oxide catalyst under 50 psi of hydrogenin ethanol and ethyl acetate in the presence of hydrogen chlorideovernight at room temperature to give 101f (380 mg, 1.74 mmol) which wasused in directly in the next step.

Example 101g 3,4,6,7,8,9-Hexahydropyrido[3,4-b]indolizin-1(2H)-one 101g

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged withmethyl 3-(2-aminoethyl)-5,6,7,8-tetrahydroindolizine-2-carboxylatehydrogen chloride salt 101f (prepared above, estimated 1.74 mmol,presuming quantitative yield), sodium ethoxide (354 mg, 5.22 mmol) andethanol (20 mL). The mixture was stirred at 55° C. for 5 h. After thattime, the reaction mixture was concentrated under reduced pressure andthe residue was partitioned between ethyl acetate (200 mL) and water(100 mL). The organic layer was separated, and the aqueous layer wasextracted with ethyl acetate (2×100 mL). The combined organic layerswere washed with brine, dried over sodium sulfate and concentrated underreduced pressure. The residue was purified by column chromatography toafford a 67% yield (220 mg) of 101g as a white solid: mp 195-197° C.; ¹HNMR (500 MHz, DMSO-d₆) δ 6.76 (s, 1H), 5.89 (s, 1H), 3.78 (t, 2H, J=6.5Hz), 3.35 (m, 2H), 2.66 (m, 4H), 1.87 (m, 2H), 1.72 (m, 2H); (APCI+) m/z191.3 (M+H)

Example 101h2-Bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]-indolizin-2(1H)-yl)benzylacetate 101h

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (60 mL), 101c(1.9 g, 6.0 mmol), 101g (400 mg, 2.0 mmol), and cesium carbonate (1.3 g,4.0 mmol). After bubbling nitrogen through the resulting mixture for 30minutes, Xantphos (120 mg, 0.2 mmol) andtris(dibenzylideneacetone)dipalladium(0) (180 mg, 0.2 mmol) were added,and the reaction mixture was heated at 100° C. for 12 h. After this timethe reaction was cooled to room temperature, partitioned between ethylacetate (40 mL) and water (40 mL), and filtered. The aqueous layer wasseparated and extracted with ethyl acetate (70 mL×3). The combinedorganic layer was washed with brine (30 mL) and dried over sodiumsulfate. The drying agent was removed by filtration and the filtrate wasconcentrated under reduced pressure. The residue was purified on flashcolumn eluting with 2:1 PE/EA to afford 101h (421 mg, 46%) as yellowsolid. MS: [M+H]⁺ 435. ¹H NMR (500 MHz, MeOD) δ 7.52-7.50 (m, 1H),7.20-7.23 (m, 1H), 6.14 (s, 1H), 5.10-5.20 (m, 2H), 4.06-4.12 (m, 1H),3.92-3.97 (m, 1H), 3.83-3.88 (m, 1H), 3.75-3.79 (m, 1H), 3.03-3.10 (m,1H), 2.94-2.99 (m, 1H), 2.75-2.83 (m, 2H), 2.00-2.05 (m, 5H), 1.83-1.88(m, 2H)

Example 101i(S)-4-Fluoro-2-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-2(1H)-yl)benzylacetate 101i

A mixture of 101h (300 mg, 0.70 mmol),(S)-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one(332 mg, 0.70 mmol), CH₃COONa (114 mg, 1.4 mmol), K₃PO₄ (368 mg, 1.4mmol), PdCl₂(dppf) (56 mg, 0.07 mmol), CH₃CN (25 mL), and H₂O (1 mL) washeated at 100° C. for 3 hours. It was then evaporated and the residuewas purified by silical-gel column eluting with methylenechloride/methanol (30:1) to afford 101i (293 mg, yield 41%) as a brownsolid. MS: (M+H)⁺ 710.

Example 101(S)-2-(5-Fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1(2H)-one101

To a solution of 101i (270 mg, 0.38 mmol) in propan-2-ol (8 mL),tetrahydrofuran (8 mL), and water (1.5 mL) was added LiOH (914 mg, 38mmol). The mixture was stirred at 30° C. for 2 h. It was evaporated andthe residue was purified by reverse-phase prep-HPLC to give 101 (127 mg,yield 50%) as a white solid. MS: (M+H)⁺ 669. ¹H NMR (500 MHz, DMSO) δ8.58 (t, J=2.5, 1H), 8.40 (s, 1H), 7.85 (d, J=3.0, 1H), 7.35-7.38 (m,2H), 7.22-7.25 (m, 2H), 7.13-7.16 (m, 1H), 6.01 (s, 1H), 4.76 (s, 1H),4.57-4.54 (m, 2H), 4.47 (t, J=6.0, 1H), 4.42 (t, J=6.5, 1H), 4.29 (s,2H), 3.98-4.04 (m, 1H), 3.89-3.94 (m, 1H), 3.78-3.83 (m, 2H), 3.67 (s,1H), 3.58 (s, 3H), 3.37-3.42 (m, 1H), 3.00-3.10 (m, 2H), 2.90-2.95 (m,2H), 2.71 (t, J=6.0, 2H), 2.52-2.55 (m, 1H), 2.28-2.35 (m, 2H), 2.18 (t,J=8.5, 1H), 1.89-1.94 (m, 2H), 1.72-1.78 (m, 2H), 0.93 (t, J=6.5, 3H)

Example 102a(S)-[4-fluoro-2-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)-piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-5-yl}phenyl]methylacetate 102a

Following the procedures as described for 101i and starting with(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trine-5-yl}-6-(tetra-methyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate (250 mg) and5-bromo-1-methyl-3-(3-methyl-5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one(218 mg), afforded 102a as a yellow solid (225 mg, 62%). LCMS: [M+H]⁺726. See FIG. 2.

Example 102(S)-5-[5-fluoro-2-(hydroxymethyl)-3(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-6-one102

Following the procedures as described for 101, hydrolysis of 102a (210mg, 0.29 mmol) with lithium hydroxide afforded 102 as a white solid (117mg, 85%). LCMS: [M+H]⁺ 684. ¹H NMR (500 MHz, CDCl₃) δ 8.61 (d, J=2.5,1H), 8.26 (s 1H), 7.99 (d, J=2.5, 1H), 7.83 (s, 1H), 7.46 (d, J=2.0,1H), 7.29-7.32 (m, 2H), 7.11 (dd, J=2.0, 8.0, 1H), 6.82 (d, J=9.0, 1H),4.62-4.73 (m, 4H), 4.31 (d, J=6.5, 2H), 4.02 (t, J=6.5, 1H), 3.71 (s,3H), 3.52-3.55 (m, 1H), 3.45-3.49 (m, 1H), 3.09 (t, J=4.5, 2H), 2.99 (t,J=4.5, 2H), 2.87 (t, J=4.5, 2H), 2.56 (dd, J=3.0, 11.0, 1H), 2.46-2.49(m, 2H), 2.19-2.25 (m, 1H), 1.96-2.01 (m, 4H), 1.00 (d, J=6.0, 3H)

Example 103a (E)-Ethyl 3-(2-Chloro-4,4-dimethylcyclopent-1-enyl)acrylate103a

The following two procedures were adapted from Organic Preparations andProcedures Int., 29 (4), 471-498. A 500-mL single neck round bottomedflask equipped with a magnetic stirrer and nitrogen inlet was chargedwith 2-chloro-4,4-dimethylcyclopent-1-enecarbaldehyde (38 g, 240 mmol)in benzene (240 mL). See FIG. 3. To the solution was addedethoxycarbonylmethylene triphenylphosphorane (84 g, 240 mmol). Themixture was stirred for 14 h. After that time, the solvent wasevaporated and the residue was triturated with hexanes (2 L) to extractthe product away from the PPh₃ by-products. The organic layer was driedover sodium sulfate and concentrated in vacuo. The residue was purifiedby column chromatography using a 100% hexane−1:1 hexane/ethyl acetategradient to afford a 37% yield (20 g) of 103a.

Example 103b Ethyl5,5-Dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylate 103b

A 250-mL single neck round bottomed flask equipped with a magneticstirrer and nitrogen inlet was charged with 103a (17 g, 74 mmol) in DMSO(100 mL). To the solution was added sodium azide (9.6 g, 150 mmol). Themixture was then heated to 75° C. and stirred for 8 h. After cooling tort, H₂O (100 mL) and CH₂Cl₂ (200 mL) were added and the organic layerwas separated. The aqueous layer was extracted with CH₂Cl₂ (50 mL). Thecombined organic layers were washed with brine, dried over sodiumsulfate and concentrated in vacuo. The residue was purified by columnchromatography using a 100% hexane-1:1 hexane/ethyl acetate gradient toafford a 37% yield (5.7 g) of 103b.

Example 103c Ethyl1-(Cyanomethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclo-penta[b]pyrrole-2-carboxylate103c

A 250-mL single neck round bottomed flask equipped with a magneticstirrer and nitrogen inlet was charged with 103b)(6.2 g, 30 mmol) in DMF(57 mL). To the solution was added NaH (80% dispersion in mineral oil,1.26 g, 42.1 mmol). The resulting mixture was stirred at rt for 90 min.After that time, bromoacetonitrile (2.94 mL, 42 mmol) was added. Themixture was stirred for 14 h. After that time, water (100 mL) and ethylacetate (200 mL) were added and the organic layer was separated. Theaqueous layer was extracted with ethyl acetate (2×50 mL). The combinedorganic layers were washed with brine, dried over sodium sulfate andconcentrated in vacuo. The residue was purified by column chromatographyto afford a 95% yield (7 g) of 103c.

Example 103d Ethyl1-(2-Aminoethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclo-penta[b]pyrrole-2-carboxylatehydrochloride 103d

A 500-mL Parr reactor bottle was purged with nitrogen and charged with10% palladium on carbon (50% wet, 2.0 g dry weight), 103c (4.5 g, 18mmol), 12% hydrochloric acid (9.2 mL, 37 mmol), ethyl acetate (80 mL)and ethanol (52 mL). The bottle was attached to a Parr hydrogenator,evacuated, charged with hydrogen gas to a pressure of 50 psi and shakenfor 6 h. After this time, the hydrogen was evacuated, and nitrogen wascharged into the bottle. Diatomaceous earth filtration agent (Celite®521, 10.0 g) was added, and the mixture was filtered through a pad ofCelite 521. The filter cake was washed with ethanol (2×50 mL), and thecombined filtrates were concentrated to dryness under reduced pressure.The crude residue 103d was carried onto the next step without furtherpurification.

Example 103e4,4-Dimethyl-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-9-one103e

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged withcrude 103d (˜18 mmol), sodium ethoxide (6.2 g, 92 mmol) and ethanol (120mL). The mixture was stirred at 55° C. over night. After that time, thereaction mixture was concentrated under reduced pressure and the residuewas partitioned between ethyl acetate (200 mL) and water (100 mL). Thesolution was filtered. The solid was washed with ethyl acetate (15 mL)to give 850 mg of desired product 103e. The organic layer was separated,and the aqueous layer was extracted with ethyl acetate (2×100 mL). Thecombined organic layers were dried over sodium sulfate and concentratedunder reduced pressure to near dryness. The solution was filtered andthe solid (1.44 g) was washed with ethyl acetate (15 mL). The combinedsolids were dried under vacuum a afford 61% yield (2.3 g) of 103e.

Example 103f2-Bromo-4-fluoro-6-(9-oxo-4,4-dimethyl-1,10diazatricyclo[6.4.0.0^(2,6)]-dodeca-2(6),7-dien-10-yl)benzylAcetate 103f

A sealed tube was equipped with a magnetic stirrer and charged with 103e(740 mg, 3.6 mmol), 2,6-dibromo-4-fluorobenzyl acetate 101c (2.4 g, 7.2mmol) and cesium carbonate (2.6 g, 7.9 mmol) in 1,4-dioxane (36 mL).After bubbling nitrogen through the solution for 30 min, Xantphos (250mg, 0.43 mmol) and tris(dibenzylideneacetone) dipalladium(0) (260 mg,0.29 mmol) were added, and the reaction mixture was heated to 100° C.for 16 h. After this time, H₂O (50 mL) and ethyl acetate (50 mL) wereadded. The aqueous layer was separated and extracted with ethyl acetate(2×50 mL). The combined organic extracts were washed with brine (100 mL)and dried over sodium sulfate. The resulting residue was purified bycolumn chromatography eluting with a gradient of 100% hexanes-100% Ethylacetate to afford a 56% yield (910 mg) of 103f.

Example 103g2-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-4-fluoro-6-(9-oxo-4,4-dimethyl-1,10diazatricyclo[6.4.0.0^(2,6)]-dodeca-2(6),7-dien-10-yl)benzylAcetate 103g

Compound 103f (450 mg, 1.0 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (635 mg, 2.5mmol), potassium acetate (393 mg, 4.0 mmol),bis(diphenylphosphino)-ferrocene]dichloropalladium(II) complex withCH₂Cl₂ (Pd Cl₂dppf:CH₂Cl₂ (1:1), 66 mg, 0.08 mmol) and 1,4-dioxane (20mL) were mixed and heated at 100° C. for 5 h. The reaction mixture wascooled to room temperature and filtered through a pad of Celite 521. Thefilter cake was washed with Ethyl Acetate (2×25 mL), and the combinedfiltrates were concentrated to dryness under reduced pressure to afford103g (quantitative yield) as black oil, which was used directly for thenext step. MS (ESI+) m/z 497.3 (M+H).

Example 103h(2S)-(2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo-[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-fluoro-6-[1-methyl-5-({5-[2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl)-methylacetate 103h

Following the procedures as described for compound 101i, reaction of(2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate (229 mg, 0.46 mmol) and(S)-5-bromo-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazine-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one(200 mg, 0.46 mmol), afforded 103h as a yellow solid (80 mg, 24%). MS:[M+H]⁺ 724.

Example 103(2S)-10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridine-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]-phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-9-one103

Following the procedures as described for 101, intermediate 103h (80 mg,0.11 mmol) was hydrolyzed with lithium hydroxide to afford 103 as ayellow solid (40 mg, 53%). LCMS: [M+H]⁺ 682. ¹H NMR (500 MHz, CDCl₃) δ8.56 (dd, J=2.0, 7.0, 1H), 7.95 (t, J=3.0, 1H), 7.82 (d, J=3.0, 1H),7.47 (t, J=3.0, 1H), 7.31 (dd, J=3.0, 9.0, 1H), 7.17-7.14 (m, 1H), 6.95(dd, J=2.5, 9.0, 1H), 6.82-6.80 (m, 2H), 4.71-4.61 (m, 4H), 4.56-4.53(m, 1H), 4.41-4.37 (m, 1H), 4.33-4.28 (m, 1H), 4.23-4.15 (m, 3H),3.91-3.86 (m, 1H), 3.70 (s, 3H), 3.55-3.44 (m, 2H), 3.08-3.06 (m, 2H),2.56-2.46 (m, 7H), 2.21-2.16 (m, 1H), 1.27 (s, 6H), 0.98-0.97 (m, 3H)

Example 104a(2R,5S)-tert-Butyl-2,5-dimethyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate104a

Following the procedures as described for compound 108a,(2R,5S)-tert-butyl 4-chloro-2,5-dimethylpiperazine-1-carboxylate (1.5 g,6.0 mmol) and 5-bromo-2-nitropyridine (1212 mg, 6.0 mol) were reacted togive 104a as a yellow solid (1500 mg, 75%). LCMS: [M+H]⁺ 337. See FIG.4.

Example 104b (2R,5S)-tert-Butyl4-(6-Aminopyridin-3-yl)-2,5-dimethylpiperazine-1-carboxylate 104b

Following the procedures as described for compound 108b, reaction of104a 1.5 g 4.46 mmol) afforded 104b as a yellow solid (1130 mg, 83%).LCMS: [M+H]⁺ 307

Example 104c (2R,5S)-tert-Butyl4-(6-(5-Bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)-2,5-dimethylpiperazine-1-carboxylate104c

Following the procedures as described for compound 108c, reaction of(2R,5S)-tert-butyl-2,5-dimethylpiperazine-1-carboxylate (332 mg, 1.08mmol) and 3,5-dibromo-1-methylpyridin-2(1H)-one (294 mg, 1.08 mmolafforded 104c as a yellow solid (402 mg, 75%). LCMS: [M+H]⁺ 492

Example 104d (2R,5S)-tert-Butyl4-(6-(5-Bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)-2,5-dimethylpiperazine-1-carboxylate104d

Following the procedures as described for compound 108d, acidichydrolysis of 104c (402 mg, 0.82 mmol) to remove the boc group afforded104d as a yellow solid (300 mg, 94%). LCMS: [M+H]⁺ 392

Example 104e5-Bromo-3-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)-pyridin-2-ylamino)-1-methylpyridin-2(1H)-one104e

Following the procedures as described for compound 108e and startingwith5-bromo-3-(5-((2S,5R)-2,5-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-pyridin-2(H)-one(300 mg, 0.77 mmol) afforded 104e as a yellow solid (320 mg, 93%). LCMS:[M+H]⁺ 448

Example 104f2,2,2-Trichloro-1-(4,5,6,7-tetrahydro-1H-indol-2-yl)ethanone 104f

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer, condenser and nitrogen inlet was purged with nitrogen andcharged with 4,5,6,7-tetrahydro-1H-indole (3.00 g, 24.8 mmol),trichloroacetyl chloride (13.5 g, 74.4 mmol) and 1,2-dichloroethane (50mL). The solution was stirred at 85° C. for 2 h. After that time, thereaction mixture was concentrated under reduced pressure to afford a100% yield (6.50 g) of 104f as a black semi-solid: ¹H NMR (500 MHz,DMSO-d₆) δ 11.94 (s, 1H), 7.05 (s, 1H), 2.62 (t, 2H, J=6.0 Hz), 2.47 (t,2H, J=6.0 Hz), 1.80 (m, 2H), 1.65 (m, 2H); MS (ESI+) m/z 266.0 (M+H)

Example 104g Ethyl 4,5,6,7-Tetrahydro-1H-indole-2-carboxylate 104g

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged with2,2,2-trichloro-1-(4,5,6,7-tetrahydro-1H-indol-2-yl)ethanone 104f (6.50g, 24.8 mmol), sodium ethoxide (17.0 mg, 0.25 mmol) and ethanol (40 mL).The solution was stirred at room temperature for 1 h. After that time,the reaction mixture was concentrated under reduced pressure. Theresidue was purified by column chromatography to afford a 100% yield(4.80 g) of ethyl 4,5,6,7-tetrahydro-1H-indole-2-carboxylate 104g as abrown solid: mp 70-72° C.; ¹H NMR (300 MHz, CDCl₃) δ 9.08 (s, 1H), 6.75(s, 1H), 4.25 (q, 2H, J=7.2 Hz), 2.65 (t, 2H, J=6.0 Hz), 2.56 (t, 2H,J=6.0 Hz), 1.85 (m, 4H), 1.28 (t, 3H, J=7.2 Hz); MS (ESI+) m/z 194.1(M+H)

Example 104h Ethyl1-(Cyanomethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 104h

A 125-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged withethyl 4,5,6,7-tetrahydro-1H-indole-2-carboxylate 104g (5.76 g, 29.8mmol) and DMF (50 mL). The solution was cooled to 0° C. using an icebath. Sodium hydride, NaH (60% dispersion in mineral oil, 1.43 g, 35.8mmol) was added. The resulting mixture was stirred at room temperaturefor 1 h. After that time, bromoacetonitrile (1.43 g, 35.8 mmol) wasadded. The mixture was stirred at room temperature for 14 h. After thattime, the reaction mixture was concentrated under reduced pressure andthe residue was partitioned between ethyl acetate (150 mL) and water(450 mL). The organic layer was separated, and the aqueous layer wasextracted with ethyl acetate (3×150 mL). The combined organic layerswere washed with brine, dried over sodium sulfate and concentrated underreduced pressure. The residue was purified by column chromatography toafford a 55% yield (3.80 g) of 104h as a yellow semi-solid: ¹H NMR (300MHz, CDCl₃) δ 6.66 (s, 1H), 5.29 (s, 2H), 4.28 (q, 2H, J=7.2 Hz), 2.62(t, 2H, J=6.3 Hz), 2.49 (t, 2H, J=6.3 Hz), 1.92 (m, 2H), 1.75 (m, 2H),1.33 (t, 3H, J=7.2 Hz); MS (ESI+) m/z 233.1 (M+H)

Example 104i Ethyl1-(2-Aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 104i

A 200-mL Parr reactor bottle was purged with nitrogen and charged with10% palladium on carbon (50% wet, 1.28 g dry weight), 104h (3.00 g, 12.9mmol), 12% hydrochloric acid (6.5 mL, 25 mmol), ethyl acetate (60 mL)and ethanol (40 mL). The bottle was attached to a Parr hydrogenator,evacuated, charged with hydrogen gas to a pressure of 50 psi and shakenfor 6 h. After this time, the hydrogen was evacuated, and nitrogen wascharged into the bottle. Celite 521 (4.0 g) was added, and the mixturewas filtered through a pad of Celite 521. The filter cake was washedwith ethanol (2×20 mL), and the combined filtrates were concentrated todryness under reduced pressure. The residue was partitioned betweenethyl acetate (150 mL) and 10% aqueous potassium carbonate (100 mL). Theorganic layer was separated, and the aqueous layer was extracted withethyl acetate (3×75 mL). The combined organic layers were dried oversodium sulfate and concentrated under reduced pressure. The residue wastriturated with ethanol (5 mL) to afford a 71% yield (1.71 g) of 104i asa white solid: mp 102-104° C.; ¹H NMR (500 MHz, DMSO-d₆) δ 6.61 (s, 1H),6.22 (br, 2H), 4.15 (m, 4H), 2.77 (m, 2H), 2.59 (t, 2H, J=6.5 Hz), 2.42(t, 2H, J=6.5 Hz), 1.70 (m, 2H), 1.62 (m, 2H), 1.23 (t, 3H, J=7.0 Hz);MS (APCI+) m/z 237.2 (M+H)

Example 104j 3,4,6,7,8,9-Hexahydropyrazino[1,2-a]indol-1(2H)-one 104j

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged with104i (1.80 g, 7.63 mmol), sodium ethoxide (1.55 g, 22.8 mmol) andethanol (50 mL). The mixture was stirred at 55° C. for 5 h. After thattime, the reaction mixture was concentrated under reduced pressure andthe residue was partitioned between ethyl acetate (200 mL) and water(100 mL). The organic layer was separated, and the aqueous layer wasextracted with ethyl acetate (2×100 mL). The combined organic layerswere washed with brine, dried over sodium sulfate and concentrated underreduced pressure. The residue was purified by column chromato-graphy toafford a 42% yield (605 mg) of 104j as a white solid: mp 207-209° C.; ¹HNMR (500 MHz, DMSO-d₆) δ 7.41 (s, 1H), 6.36 (s, 1H), 3.84 (t, 2H, J=6.0Hz), 3.42 (m, 2H), 2.51 (t, 2H, J=6.0 Hz), 2.42 (t, 2H, J=6.0 Hz), 1.76(m, 2H), 1.65 (m, 2H); (APCI+) m/z 191.3 (M+H)

Example 104k 2,6-Dibromo-4-fluorobenzaldehyde 104k

To a solution of 1,3-dibromo-5-fluoro-2-iodobenzene (50 g, 132 mmol) inanhydrous toluene (300 mL) cooled at −35° C. was added a solution ofisopropyl-magnesium chloride (84 mL, 171 mmol, 2.0M in Et₂O) over 30minutes while maintaining the internal temperature below −25° C. A clearbrown solution was obtained and the stirring was continued for 1.5 h at−25° C. Then anhydrous DMF (34 mL, 436 mmol) was added over a period of30 minutes. The reaction mixture was warmed to 10° C. (room temperature)over 1 h and stirred at this temperature for 1.5 h (hours). It was thenquenched with 3.0M HCl and followed by the addition of ethyl acetate.The organic layer was separated and evaporated under reduced pressure.The residue was purified by silica-gel column chromatography elutingwith petroleum ether/ethyl acetate (from 50:1 to 20:1) to give 104k as awhite solid (20 g, 54%). ¹H NMR (500 MHz, CDCl₃) δ 10.23 (s, 1H), 7.48(d, J=7.5, 2H).

Example 104l (2,6-Dibromo-4-fluorophenyl)methanol 104l

To a solution of 104k (20 g, 71 mmol) in EtOH (500 mL) was added NaBH₄(10 g, 284 mmol). The mixture was stirred at room temperature (10° C.)for 4 h and TLC showed the starting material had disappeared. Thereaction was quenched by aqueous HCl solution (150 mL, 1M) andevaporated in vacuo until most of EtOH was distilled. The residue wasextracted by ethyl acetate (500 mL×3). The organic layers were combined,dried with Na₂SO₄, and evaporated in vacuo. The residue was purified bysilica-gel column chromatography eluting with petroleum ether/ethylacetate (from 50:1 to 20:1) to give 1041 as a white solid (15 g, 75%).MS: [M−OH]⁺ 267. ¹H NMR (500 MHz, DMSO-d₆) δ 7.68 (d, J=8.5, 2H), 5.23(s, 1H), 4.71 (s, 2H).

Example 104m 2,6-Dibromo-4-fluorobenzyl Acetate 104m

To a solution of (2,6-dibromo-4-fluorophenyl)methanol (1041) (20 g, 71mmol) in CH₂Cl₂ (500 mL) at 0° C. was added pyridine (8.4 g, 107 mmol)and acetyl chloride (8.3 g, 107 mmol). The mixture was stirred at roomtemperature for 5 h. TLC showed the start material disappeared. Thereaction was evaporated in vacuum and the residue was purified bysilica-gel column chromatography eluting with petroleum ether/ethylacetate (from 50:1 to 20:1) to give 104m as a white solid (20 g, 87%).MS: [M-Oac]⁺ 267. ¹H NMR (500 MHz, CDCl₃) δ 7.36 (d, J=7.5, 2H), 5.38(s, 2H), 2.10 (s, 3H)

Example 104n2-Bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 104n

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 104j (3.8 g, 20 mmol), 104m (20 g, 60 mmol),XantPhos (1.2 g, 2 mmol), tris(dibenzylideneacetone)dipalladium(0) (1.8g, 2 mmol), Cs₂CO₃ (16 g, 50 mmol), and 1,4-dioxane (120 mL). The systemwas evacuated and then refilled with N₂. A reflux condenser was attachedto the flask, and the reaction mixture was heated at 100° C. for 16 h.Then, the mixture was cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure and the resultingresidue was purified by flash column chromatography eluting with 5:1petroleum ether/ethyl acetate to afford 104n as a white solid (5.2 g,60%). MS: [M+H]⁺ 435. ¹H NMR (500 MHz, DMSO-d₆) δ 7.70 (dd, J=3, 1H),7.48 (dd, J=3, 1H), 6.52 (s, 1H), 5.01 (m, 2H), 4.18 (m, 2H), 4.02 (m,1H), 3.73 (m, 1H), 2.60 (m, 2H), 2.45 (m, 2H), 1.98 (s, 3H), 1.77 (m,2H), 1.68 (m, 2H).

Example 104o4-Fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 104o

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 104n (3.8 g, 8.65 mmol), (PinB)₂ (11 g, 43.25mmol), Pd(dppf)Cl₂ (0.4 g, 0.5 mmol), KOAc (2.5 g, 26 mmol), and1,4-dioxane (150 mL). The system was evacuated and then refilled withN₂. A reflux condenser was attached to the flask and the reactionmixture was heated at 100° C. for 15 h. Then, the mixture was cooled toroom temperature and filtered. The filtrate was concentrated underreduced pressure and the resulting residue was purified by flash columnchromatography eluting with 5:1 petroleum ether/ethyl acetate to afford104o as a yellow solid (3.2 g, 77%). MS: [M+H]⁺ 483.

Example 104p2-(5-(5-((2S,5R)-2,5-Dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylAcetate 104p

Following the procedures as described for compound 108f, reaction of104e (268 mg, 0.60 mmol) and 104o (289 mg, 0.60 mmol) afforded 104p as ayellow solid (300 mg, 85%). LCMS: [M+H]⁺ 724

Example 1042-(3-(5-(5-((2S,5R)-2,5-Dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one104

Following the procedures in Example 101i, hydrolysis of the acetateester of 104p (288 mg, 0.40 mmol) with lithium hydroxide afforded 104 asa white solid (80 mg, 25%). LCMS: [M+H]⁺ 682. ¹H NMR (500 MHz, CDCl₃) δ8.60 (d, J=2.0, 1H), 8.02 (d, J=2.5, 1H), 7.87 (d, J=1.5, 1H), 7.49-7.48(m, 1H), 7.37 (d, J=9.0, 1H), 7.16 (d, J=9.0, 1H), 6.96 (d, J=8.5, 1H),6.87 (s, 1H), 6.81 (d, J=9.0, 1H), 4.77-4.61 (m, 4H), 4.54 (d, J=11.5,1H), 4.39-4.31 (m, 2H), 4.19-4.15 (m, 3H), 3.92-3.91 (m, 1H), 3.77-3.74(m, 1H), 3.71 (s, 3H), 3.18 (s, 1H), 2.92-2.89 (m, 1H), 2.73-2.70 (m,2H), 2.61-2.56 (m, 4H), 2.48 (s, 1H), 1.98-1.79 (m, 5H), 0.91-0.89 (m,6H)

Example 105a(S)-2-(5-(5-(2-Ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 105a

A sealed tube equipped with a magnetic stirrer was charged with(S)-5-bromo-3-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one(203 mg, 0.45 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate (216 mg, 0.45 mmol), Pd(dppf)Cl₂ (18 mg, 0.0225 mmol), NaOAc (74mg, 0.90 mmol), K₃PO₄ (191 mg, 0.90 mmol), and acetonitrile (3 mL). SeeFIG. 5. After three cycles of vacuum/argon flush, the mixture was heatedat 100° C. for 1 h. It was then filtered and the filtrate was evaporatedin vacuo. The residue was purified by silica gel column chromatographyeluting with dichloromethane/methanol (25:1, UV) to afford 105a (220 mg,87%) as a brown solid. LCMS: [M+H]⁺ 724

Example 105(S)-2-(3-(5-(5-(2-Ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one105

A mixture of 105a (220 mg, 0.30 mmol) and LiOH (72 mg, 3.0 mmol) in^(i)PrOH/THF (1:1, 4 mL) and H₂O (1 mL) was stirred at 30° C. for 1 h.The mixture was evaporated in vacuo and the residue was extracted withEtOAc (10 mL×2). The combined EtOAc extract was concentrated underreduced pressure and the residue was purified by reverse-phase prep-HPLCto afford 105 (54 mg, 25%) as a white solid. LCMS: [M+H]⁺ 682. ¹H NMR(500 MHz, CDCl₃) δ 8.54 (t, J=2.5, 1H), 7.91 (s, 1H), 7.80 (s, 1H), 7.47(d, J=1.5, 1H), 7.26-7.24 (m, 1H), 7.18-7.15 (m, 1H), 6.95 (dd, J=2.5,9.0, 1H), 6.87 (s, 1H), 6.81 (d, J=7.5, 1H), 4.72-4.61 (m, 4H), 4.54 (d,J=11.5, 1H), 4.33-4.30 (m, 2H), 4.20-4.14 (m, 3H), 3.92-3.90 (m, 1H),3.71 (s, 3H), 3.53 (t, J=5.5, 1H), 3.31 (s, 1H), 3.12 (s, 2H), 2.61-2.56(m, 5H), 2.44 (s, 2H), 2.35 (s, 1H), 1.91-1.89 (m, 2H), 1.80-1.79 (m,2H), 1.67 (s, 1H), 1.43-1.37 (m, 1H), 0.82 (t, J=5.5, 3H)

Example 106a(S)-4-fluoro-2-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 106a

A sealed tube was charged with the mixture of4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexa-hydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate ((337 mg, 0.7 mmol),(S)-5-bromo-1-methyl-3-(3-methyl-5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one(303 mg, 0.7 mmol), Pd(dppf)Cl₂ (33 mg, 0.04 mmol), K₃PO₄.3H₂O(372 mg,1.4 mmol), and NaOAc (115 mg, 1.4 mmol) in CH₃CN (20 mL). See FIG. 6.The system was evacuated and refilled with N₂. The reaction mixture washeated at 110° C. for 2 h. It was then cooled to room temperature andfiltered. The filtrate was concentrated under reduced pressure and theresulting residue was purified by flash column chromatography elutingwith 30:1 DCM/MeOH to afford 106a as a yellow solid (258 mg, 52%). LCMS:[M+H]⁺ 710

Example 106(S)-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one106

At room temperature, to a solution of 106a (153 mg 0.22 mmol) inTHF/iPA/H₂O (6 mL/6 mL/2 mL) was added LiOH (70 mg, 2.9 mmol) whilestirring. This mixture was stirred for 0.5 h. Then, H₂O (20 mL) wasadded and the mixture was extracted with EA (30 mL×3). The combinedorganic layer was dried with Na₂SO₄ and concentrated to give a yellowsolid, which was further purified by reverse-phase prep-HPLC to afford106 as a white solid (60 mg, 42% yield). LCMS: [M+H]⁺ 668. ¹H NMR (500MHz, CDCl₃) δ 8.57 (dd, J=2.0, 7.0, 1H), 7.97 (s, 1H), 7.85 (s, 1H),7.48 (t, J=2.5, 1H), 7.33 (d, J=7.0, 1H), 7.16 (d, J=8.0, 1H), 6.96 (dd,J=2.5, 9.0, 1H), 6.87 (s, 1H), 6.83 (d, J=9.0, 1H), 4.69-4.72 (m, 4H),4.55 (d, J=11.5, 1H), 4.29-4.38 (m, 2H), 4.14-4.20 (m, 3H), 3.90-3.93(m, 1H), 3.71 (s, 3H), 3.57 (s, 1H), 3.48 (s, 1H), 3.09-3.14 (m, 2H),2.52-2.61 (m, 7H), 2.23 (s, 1H), 1.88-1.91 (m, 2H), 1.79-1.81 (m, 2H),0.99 (d, J=5.0, 3H)

Example 107a (S)-tert-Butyl2-Methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 107a

Following the procedures as described for compound 108a, reaction of5-bromo-2-nitropyridine (1.5 g) and (S)-tert-butyl2-methylpiperazine-1-carboxylate (2.3 g), gave 107a as a yellow solid(1.5 g, 40%). MS: [M+H]⁺ 323. See FIG. 7.

Example 107b (S)-tert-Butyl4-(6-Aminopyridin-3-yl)-2-methyl-piperazine-1-carboxylate 107b

Following the procedures as described for compound 108b, reduction of107a (4.0 g) gave 107b as a yellow solid (1.23 g, 97%). MS: [M+H]⁺ 293

Example 107c (S)-tert-Butyl4-(6-(5-Bromo-1-methyl-2-oxo-1,2-dihydro-pyridin-3-ylamino)pyridin-3-yl)-2-methylpiperazine-1-carboxylate107c

Following the procedures as described for compound 108c, reaction of3,5-dibromo-1-methylpyridin-2(1H)-one (83 mg) and 107b (90 mg), gave107c as a yellow solid (120 mg, 81%). MS: [M+H]⁺ 480

Example 107d(S)-5-bromo-1-methyl-3-(5-(3-methylpiperazin-1-yl)-pyridin-2-yl-amino)pyridin-2(1H)-one107d

Following the procedures as described for compound 108d, acidichydrolysis of the boc group of 107c (120 mg) gave 107d as a yellow solid(100 mg, 90%). MS: [M+H]⁺ 380

Example 107e(S)-5-bromo-3-(5-(3,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one107e

Following the procedures as described for compound 108e, methylation of107d (100 mg) gave 107e as a yellow solid (60 mg, 59%). MS: [M+H]⁺ 394

Example 107f(S)-2-(5-(5-(3,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino-[1,2-a]indol-2(1H)-yl)benzylacetate 107f

Following the procedures as described for compound 108f, reaction of4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate (74 mg) and 107e (60 mg) gave 107f as a yellow solid (60 mg,59%). LCMS: [M+H]⁺ 668

Example 107(S)-2-(3-(5-(5-(3,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one107

Following the procedures in Example 108, hydrolysis of 107f (60 mg) withlithium hydroxide gave 107 as a white solid (20 mg, 36%). LCMS: [M+H]⁺626. ¹H NMR (500 MHz, CDCl₃) δ: 8.55 (s, 1H), 7.90 (d, J=2.5, 1H), 7.77(s, 1H), 7.46 (d, J=2.5, 1H), 7.26 (dd, J=3.0, 9.5, 1H), 7.17 (dd,J=3.0, 9.5, 1H), 6.95 (dd, J=3.0, 9.0, 1H), 6.87 (s, 1H), 6.81 (d,J=9.0, 1H), 4.55 (d, J=10.5, 1H), 4.31-4.36 (m, 2H), 4.14-4.20 (m, 3H),3.90-3.92 (m, 1H), 3.70 (s, 3H), 3.38 (d, J=11, 1H), 3.32 (d, J=11.5,1H), 2.94 (s, 2H), 2.56-2.61 (m, 5H), 2.49 (s, 1H), 2.38 (s, 4H),1.89-1.91 (m, 2H), 1.79-1.80 (m, 2H), 1.17 (d, J=5.0, 3H)

Example 108a (R)-tert-Butyl2-Methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 108a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with methylsulfinylmethane(50 mL), 5-bromo-2-nitropyridine (2.2 g, 11 mmol), (R)-tert-butyl2-methylpiperazine-1-carboxylate (2.2 g, 11 mmol), and potassiumcarbonate (3.08 g, 22 mmol). See FIG. 8. The system was subjected tothree cycles of vacuum/nitrogen flush and heated at 65° C. for 15 h. Itwas then cooled to room temperature, and partitioned between ethylacetate (100 mL) and water (20 mL). The aqueous layer was separated andextracted with ethyl acetate (50 mL×2). The combined organic layer waswashed with brine (50 mL) and dried over anhydrous sodium sulfate. Thedrying agent was removed by filtration and the filtrate was concentratedunder reduced pressure. The residue was purified on flash columnchromatography eluting with 2:1 (UV) petroleum/ethyl acetate to afford108a as a yellow solid (1.75 g, 50%). LCMS: [M+H]⁺ 323

Example 108b (R)-tert-Butyl4-(6-aminopyridin-3-yl)-2-methylpiperazine-1-carboxylate 108b

To a mixture of 108a (1.0 g, 3.1 mmol) in methanol (15 mL) was added 10%palladium carbon (100 mg). The mixture was stirred at room temperatureunder the atmosphere of hydrogen for overnight. At the end of reaction,it was filtered and the filtrate was concentrated under reducedpressure. The residue was purified by silica gel column chromatographyeluting with DCM/MeOH (10:1, UV) to afford 108b as a brown solid (800mg, 88%). LCMS: [M+H]⁺ 293

Example 108c (R)-tert-Butyl4-(6-(5-Bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridine-3-yl)-2-methylpiperazine-1-carboxylate 108c

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (15 mL),108b (800 mg, 2.7 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (720 mg,2.7 mmol), and cesium carbonate (1.76 g, 5.4 mmol). After bubblingnitrogen through the suspension for 3 minutes, Xantphos (78 mg, 0.14mmol) and tris(dibenzylideneacetone)dipalladium(0) (128 mg, 0.14 mmol)were added. The system was subject to three cycles of vacuum/argon flashand heated at reflux for 3 h. It was then cooled to room temperature andfiltered. The filtrate was partitioned between ethyl acetate (30 mL) andwater (30 mL). The aqueous layer was separated and extracted with ethylacetate (50 mL×2). The combined organic layer was washed with brine (50mL) and dried over anhydrous sodium sulfate. The drying agent wasremoved by filtration and the filtrate was concentrated under reducedpressure. The residue was purified on flash column chromatographyeluting with 10:1 dichloromethane/methanol to afford 108c as a yellowsolid (624 mg, 47%). LCMS: [M+H]⁺ 480

Example 108d(R)-5-bromo-1-methyl-3-(5-(3-methylpiperazin-1-yl)pyridin-2-ylamino)pyridine-2(1H)-one108d

To a mixture of 108c (624 mg, 1.3 mmol) in dichloromethane (8 mL) wasadded trifluoroacetic acid dropwise (300 mg, 2.6 mmol) at roomtemperature. Then the mixture was stirred for overnight. 2.0 N NaOH wasadded to adjust pH to greater than 10. Then the mixture was extractedwith ethyl acetate (50 mL×2). The combined organic layer was washed withbrine (50 mL), dried over anhydrous sodium sulfate, and concentratedunder reduced pressure to afford 108d as a brown solid (300 mg, 61%).LCMS: [M+H]⁺ 380

Example 108e(R)-5-Bromo-3-(5-(3,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one108e

To a solution of 108d (300 mg, 0.8 mmol) in methanol (8 mL) was addedtwo drops of acetic acid, 30% formaldehyde solution (0.5 mL), and sodiumtriacetoxyborohydride (354 mg, 1.6 mmol) at room temperature. Then themixture was stirred at room temperature for 1 h. At the end of reaction,water (10 mL) was added and the mixture was extracted with ethyl acetate(20 mL×2). The combined organic layer was dried over anhydrous sodiumsulfate and concentrated under reduced pressure to afford 108e as abrown solid (280 mg, 90%). LCMS: [M+H]⁺ 392

Example 108f(R)-2-(5-(5-(3,4-Dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 108f

A sealed tube equipped with a magnetic stirrer was charged with 108e(280 mg, 0.71 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate (344 mg, 0.71 mmol), PdCl₂(dppf) (58 mg, 0.071 mmol), 1.0 MNaOAc (2.0 equiv), 1.0 M K₃PO₄ (2.0 equiv), and dioxane (5 mL). Afterthree cycles of vacuum/argon flush, the mixture was heated at 110° C.for 2 h. It was then filtered and the filtrate was evaporated in vacuo.The residue was purified by silica gel column chromatography elutingwith dichloromethane/methanol (10:1, V/V) to afford 108f (250 mg, 58%)as a white solid. LCMS: [M+H]⁺ 668

Example 108(R)-2-(3-(5-(5-(3,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)-phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one108

A mixture of 108f (250 mg, 0.375 mmol) and LiOH.H₂O (98 mg, 2.0 mmol) in^(i)PrOH/THF (1:1, 3 mL) and H₂O (1 mL) was stirred at 30° C. for 2 h.The mixture was evaporated in vacuo and the residue was extracted withEtOAc (10 mL×2). The combined EtOAc extract was concentrated underreduced pressure and the residue was purified with reverse-phaseprep-HPLC to afford 108 (62 mg, 26%) as a white solid. LCMS: [M+H]⁺ 626.¹H NMR (500 MHz, CDCl₃) δ 8.53 (s, 1H), 7.89 (d, J=2.5, 1H), 7.76 (s,1H), 7.45 (d, J=2, 1H), 7.26 (s, 1H), 7.14-7.16 (m, 1H), 6.92-6.94 (m,1H), 6.86 (s, 1H), 6.79 (d, J=9, 1H), 4.52-4.54 (m, 1H), 4.30-4.32 (m,2H), 4.14-1.16 (m, 3H), 3.90-3.91 (m, 1H), 3.69 (s, 3H), 3.34-3.35 (m,2H), 2.91-2.92 (m, 2H), 2.60-2.38 (m, 10H), 1.83-1.85 (m, 2H), 1.78-1.79(m, 2H), 1.17 (s, 3H)

Example 109a (R)-tert-Butyl3-Methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 109a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (60 mL),5-bromo-2-nitropyridine (2.0 g, 10.0 mmol), (R)-tert-butyl3-methylpiperazine-1-carboxylate (2.0 g, 10.0 mmol), and cesiumcarbonate (6.5 g, 20 mmol). See FIG. 9. After bubbling nitrogen throughthe resulting mixture for 30 minutes, XantPhos (579 mg, 1.0 mmol) andtris(di-benzylideneacetone)dipalladium(0) (915 mg, 1.0 mmol) were added,and the reaction mixture was heated at 100° C. for 15 h. After this timethe reaction was cooled to room temperature and filtered. The filtratewas partitioned between ethyl acetate (100 mL) and water (100 mL). Theaqueous layer was separated and extracted with ethyl acetate (150 mL×3).The combined organic layer was washed with brine (50 mL) and dried oversodium sulfate. The drying agent was removed by filtration and thefiltrate was concentrated under reduced pressure. The residue waspurified on flash column eluting with 30:1 DCM/MeOH to afford 109a (1.6g, 44%) as a yellow solid. MS: [M+H]⁺ 323. ¹H NMR (500 MHz, DMSO) δ 8.21(d, J=3.5, 1H), 8.18 (d, J=9.0, 1H), 7.43-7.45 (m, 1H), 4.33 (s, 1H),3.92-3.99 (m, 1H), 3.80 (d, J=12.5, 2H), 3.06-3.23 (m, 3H), 1.43 (s,9H), 1.09 (d, J=6.5, 3H).

Example 109b (R)-tert-Butyl4-(6-Aminopyridin-3-yl)-3-methylpiperazine-1-carboxylate 109b

A 500-mL flask was purged with nitrogen and charged with 109a (1.5 g,4.6 mmol), 10% palladium on carbon (50% wet, 200 mg), and methanol (70mL). It was evacuated, charged with hydrogen gas, and stirred at roomtemperature for 10 h. The hydrogen was then evacuated and nitrogen wascharged into the flask. The catalyst was removed by filtration through apad of Celite and the filtrate was concentrated under reduced pressureto afford 109b (1.1 g, 81%) as a brown solid. MS: [M+H]⁺ 293

Example 109c(R)-tert-Butyl-4-(6-(5-Bromo-1-methyl-2-oxo-1,2-dihydro-pyridin-3-ylamino)pyridin-3-yl)-3-methylpiperazine-1-carboxylate109c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (50 mL), 109b(1.0 g, 3.4 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (2.7 g, 10.2mmol), and cesium carbonate (2.2 g, 6.8 mmol). After bubbling nitrogenthrough the resulting mixture for 30 minutes, XantPhos (198 mg, 0.34mmol) and tris(dibenzylideneacetone)-dipalladium(0) (313 mg, 0.34 mmol)were added, and the reaction mixture was heated at 100° C. for 5 h.After this time the reaction was cooled to room temperature andfiltered. The filtrate was partitioned between ethyl acetate (100 mL)and water (100 mL). The aqueous layer was separated and extracted withethyl acetate (100 mL×3). The combined organic layer was washed withbrine (50 mL) and dried over sodium sulfate. The drying agent wasremoved by filtration and the filtrate was concentrated under reducedpressure. The residue was purified on flash column eluting with 30:1DCM/MeOH to afford 109c as a yellow solid (1.1 g, 63%). MS: [M+H]⁺ 478.

Example 109d(R)-5-Bromo-1-methyl-3-(5-(2-methylpiperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one109d

A mixture of 109c (600 mg, 1.26 mmol) in methanol (20 mL) was addedHCl/dioxane (4.0 M, 4 mL). The reaction mixture was stirred at roomtemperature for 4 h. It was then concentrated at reduced pressure. Theresidue was basified with aqueous 1.0 M NaOH and extracted with DCM. Thecombined organic layer was washed with H₂O and concentrated underreduced pressure to afford 109d (450 mg, 95%) as yellow solid. MS:[M+H]⁺ 378.

Example 109e(R)-5-Bromo-3-(5-(2,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one109e

A mixture of 109d (500 mg, 1.3 mmol), and 30% formaldehyde (6.5 mmol) inmethanol/HOAc (30 mL/3 mL) was stirred at room temperature for 5minutes, followed by the addition of NaBH₃CN (120 mg, 1.9 mmol). Themixture was stirred at room temperature for 4 h. It was cooled to roomtemperature and H₂O (20 mL) was added. The mixture was extracted withDCM (50 mL) three times. The combined organic layer was concentratedunder reduced pressure and the residue was purified by columnchromatography eluting with 30:1 DCM/methanol to afford 109e as a yellowsolid (473 mg, 83%). MS: [M+H]⁺ 392.

Example 109f(R)-2-(5-(5-(2,4-Dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 109f

A mixture of 109e (400 mg, 1.0 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate (490 mg, 1.0 mmol), PdCl₂(dppf) (80 mg, 0.1 mmol), 2.0 M Na₂CO₃(2.0 equiv) in DMF (4 mL) was heated at 100° C. for 2 h. Brine was addedand the mixture was extracted with EA (50 mL) three times. The combinedorganic layer was concentrated under reduced pressure. The residue waspurified by flash column chromatography eluting with 30:1 DCM/MeOH toafford 109f as a brown solid (354 mg, 52%). LCMS: [M+H]⁺ 668

Example 109(R)-2-(3-(5-(5-(2,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one109

Following the procedures as described for compound 108, hydrolysis of109f (337 mg, 0.5 mmol) with lithium hydroxide gave 109 as a yellowsolid (152 mg, 48%). LCMS: (M+H)⁺ 626. ¹H NMR (500 MHz, DMSO) δ 8.57 (t,J=2.5, 1H), 8.39 (s, 1H), 7.83 (d, J=3.0, 1H), 7.31-7.36 (m, 3H), 7.23(d, J=9.0, 1H), 7.17-7.20 (m, 1H), 6.53 (s, 1H), 4.86-4.88 (m, 1H), 4.32(d, J=4.5, 2H), 4.09-4.20 (m, 3H), 3.87-3.91 (m, 1H), 3.66 (s, 1H), 3.59(s, 3H), 3.04-3.08 (m, 1H), 2.90-2.94 (m, 1H), 2.57-2.65 (m, 3H), 2.47(t, J=5.5, 2H), 2.35-2.42 (m, 2H), 2.19-2.21 (m, 1H), 2.18 (s, 3H), 1.79(t, J=6.0, 2H), 1.66-1.69 (m, 2H), 0.91 (d, J=6.0, 3H)

Example 110a(S)-5-Bromo-3-(5-(2,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one110a

Following the procedures as described for compound 109e,(S)-5-bromo-1-methyl-3-(5-(2-methylpiperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one(377 mg, 1 mmol) was methylated by reductive formylation to give 110a asa white solid (294 mg, 75%). LCMS: [M+H]⁺ 393. See FIG. 10.

Example 110b(S)-2-(5-(5-(2,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 110b

Following the procedures as described for compound 109f, 110a (391 mg, 1mmol) and4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate (482 mg, 1 mmol) were coupled by Suzuki reaction to give 110b asa white solid (334 mg, 50%). LCMS: [M+H]⁺ 668

Example 110(S)-2-(3-(5-(5-(2,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one110

Following the procedures in Example 109, hydrolysis of the acetate esterof 110b (667 mg, 1.0 mmol), gave 110 as a white solid (75 mg, 12%).LCMS: [M+H]⁺ 626. ¹H NMR (500 MHz, CDCl₃) δ 8.57 (d, J=8, 1H), 7.96 (s,1H), 7.82 (s, 1H), 7.47 (d, J=1.5, 1H), 7.33 (s, 1H), 7.15-7.16 (m, 1H),6.94-6.96 (m, 1H), 6.86 (s, 1H), 6.80 (d, J=8.5, 1H), 4.52-4.54 (m, 1H),4.31-4.33 (m, 2H), 4.18-4.19 (m, 3H), 3.90-3.91 (m, 2H), 3.70 (s, 3H),3.49-3.50 (m, 1H), 3.06-3.07 (m, 2H), 2.58-2.60 (m, 7H), 2.34 (s, 3H),1.88-1.89 (m, 2H), 1.78-1.79 (m, 2H), 0.96 (s, 3H)

Example 111a 1-tert-Butyl 2-Methyl 4-benzylpiperazine-1,2-dicarboxylate111a

To a dry 100 ml one necked round bottom flask equipped with a stirringbar was added 1-tert-butyl 2-methyl piperazine-1,2-dicarboxylate (5 g,20.5 mmol) under N₂ (FIG. 1). Anhydrous acetonitrile (60 mL) was addedfollowed by the additions of BnBr (2.7 mL, 22.5 mmol) and triethylamine(8.5 ml, 61.5 mmol). A condenser was then put on to the flask and thereaction mixture was heated at 71° C. for 45 minutes. The reactionmixture was allowed to come to room temperature and concentrated underreduced pressure. It was then diluted with dichloromethane and washedwith water and brine. The organic layer was dried over Na₂SO₄, filteredand concentrated under reduced pressure. The crude compound was purifiedwith flash column (PE: EA=8:1) to yield 4.5 g (66%) of 111a. MS: [M+H]+:335

Example 111b (4-benzyl-1-methylpiperazin-2-yl)methanol 111b

Compound 111a (1 g, 2.99 mmol) was dissolved in 100 mL of anhydroustetrahydrofuran and lithium aluminum hydride (342 mg, 8.98 mmol) wasadded carefully at 0° C. and the mixture was stirred for 30 min. Then,it was refluxed for 3 h and the reaction mixture was poured onto iceportion-wise. It was then filtered and the filtrate was evaporated invacuo. After addition of 100 mL brine, it was extracted withdichloromethane (100 mL×3). The combined organic layer was dried overNa₂SO₄, filtered and concentrated to afford 111b as yellow oil (0.6 g,yield 91%)

Example 111c 4-benzyl-2-(fluoromethyl)-1-methylpiperazine 111C

To an ice cooled solution of N,N′-dimethylaminosulfur trifluoride (10.8ml, 81.8 mmol) in methylene chloride under N₂ was added 111b (9 g, 40.9mmol) in methylene chloride dropwise. The yellow solution was stirred at0° C. for 1 hour and warmed to room temperature and stirred for 15hours. The reaction was diluted with NaHCO₃, and the organic layer wasseparated and dried over Na₂SO₄. The crude product was purified onsilica gel (DCM:MeOH=50:1) to afford 111c as a yellow oil (3 g, yield33%). MS: [M+H]+: 223

Example 111d 2-(Fluoromethyl)-1-methylpiperazine 111d

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 111c (3 g, 13.5 mmol) and MeOH (80 mL), and theresulting mixture was added Pd/C (10%) (300 mg). The reaction mixturewas stirred under hydrogen gas (H₂) for 15 h. After the reaction wasfinished, it was filtered and concentrated to afford 111d as a yellowoil (1.6 g, yield 90%).

Example 111e 2-(Fluoromethyl)-1-methyl-4-(6-nitropyridin-3-yl)piperazine111e

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 111d (1.6 g, 12.1 mmol),5-bromo-2-nitropyridine (3.7 g, 18.2 mmol) and cesium carbonate (9.9 g,30.2 mmol). After bubbling nitrogen through the resulting solution for30 min, xantphos (700 mg, 0.12 mmol) andtris(dibenzyl-ideneacetone)dipalladium(0) (550 mg, 0.06 mmol) wereadded, and the reaction mixture was heated at reflux for 15 h. Afterthis time the reaction was cooled to room temperature, filtered andconcentrated to afford a black solid as the crude product. Then it waspurified on silica gel (DCM:MeOH=100:1) to afford 111e as a yellow solid(2.6 g, yield 76%). ¹H NMR (500 MHz, MeOD) δ 8.20 (dd, J=12.5 Hz, 2H),7.51 (dd, J=9.0 Hz, 1H), 4.74-4.54 (m, 2H), 4.03-3.92 (m, 2H), 3.20 (m,1H), 3.09-2.99 (m, 2H), 2.50 (m, 2H), 2.46 (s, 3H),

Example 111f 5-(3-(Fluoromethyl)-4-methylpiperazin-1-yl)pyridin-2-amine111f

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 111e (2.6 g, 10.2 mmol) and MeOH (50 mL), andthe resulting mixture was added Pd/C (10%) (260 mg). The reactionmixture was stirred under H₂ for 15 h. After the reaction was finished,it was filtered and concentrated to afford 111f, which was used in thenext step without purification.

Example 111g5-Bromo-3-(5-(3-(fluoromethyl)-4-methylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one111g

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (60 mL), 111e(crude, 14.1 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (4.5 g, 16.9mmol) and cesium carbonate (11.5 g, 35.2 mmol). After bubbling nitrogenthrough the resulting solution for 30 min, Xantphos (820 mg, 1.41 mmol)and tris(dibenzylideneacetone)dipalladium(0) (645 mg, 0.7 mmol) wereadded, and the reaction mixture was heated at reflux for 15 h. Afterthis time the reaction was cooled to room temperature, filtered andconcentrated to afford a black solid as the crude product. Then it waspurified on silica gel (DCM:MeOH=100:1 to 50:1) to afford 111g as ayellow solid (3.1 g, yield 50%).

Example 111h4-Fluoro-2-(5-(5-(3-(fluoromethyl)-4-methylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexa-hydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 111h

A mixture of 111g (1 g, 2.4 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate (1.3 g, 2.68 mmol), PdCl₂(dppf) (190 mg, 0.24 mmol), K₃PO₄ (1 g,4.8 mmol), and NaOAc (390 mg, 4.8 mmol) in MeCN (15 mL) and H₂O (1.5 mL)was heated at 110° C. for 3 h. The solvent was evaporated in vacuo. Theresidue was purified on silica gel column (DCM: MeOH=50:1) to give 111h(0.8 g, yield 45%).

Example 1112-(5-Fluoro-3-(5-(5-(3-(fluoromethyl)-4-methylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one111

A mixture of 111h (750 mg, 1.09 mmol) and LiOH hydrate (2.3 g, 55 mmol)in iPrOH (10 mL), THF (10 mL) and H₂O (10 mL) was stirred at 30° C. for1 h. The mixture was evaporated in vacuo, and the residue was extractedwith DCM (3×30 mL). The combined extracts were concentrated underreduced pressure. And the residue was purified on silica gel column(DCM: MeOH=50:1) to give 111 as a yellow solid (700 mg, 93%). MS: [M+H]⁺644. ¹H NMR (500 MHz, MeOD) δ 8.54 (d, J=2.0 Hz, 1H), 7.93 (d, J=1.5 Hz,1H), 7.41 (m, 1H), 7.33 (d, J=2.0 Hz, 1H), 7.21 (d, J=9.0 Hz, 2H), 7.02(d, J=9.0 Hz, 1H), 6.72 (s, 1H), 4.67-4.46 (m, 4H), 4.19 (s, 3H), 4.02(m, 1H), 3.70 (s, 3H), 3.51 (d, J=11.5 Hz, 1H), 3.42 (d, J=11. Hz, 1H),3.36 (s, 1H), 2.96 (d, J=11.5 Hz, 1H), 2.85 (m, 1H), 2.73-2.50 (m, 7H),2.48 (s, 3H), 1.88 (m, 2H), 1.78 (m, 2H)

Example 112a N,N-Dibromobenzenesulfonamide 112a

Benzensulfonamide (115g, 731.4 mmol), KOH (82.8 g, 1.48 mol) and water(500 ml) were placed in a 1000-mL three-neck flask (FIG. 12). Thenbromine (230 g, 1.48 mol) was added dropwise with vigorous stirring. Theresulting precipitate was filtered, washed with water and filtered togive 112a as a yellow powder (207g, 90%)

Example 112b (S)-ethyl2-bromo-3-(N—((R)-2-bromo-3-ethoxy-3-oxopropyl)phenyl-sulfonamido)propanoate112b

To a solution of 112a (207g, 658.26 mmol) in DCM (500 ml) was addedethyl acrylate (331.2 g, 3.29 mol). The mixture was stirred to refluxand the mercury-arc lamp was opened to ensure the reaction taking placein the light of mercury-arc for 4 h. Then the reaction mixture waspurified through a silica gel column eluting with PE:EA=15:1 to 10:1 togive 112b as a white solid (20 g, 6%). MS: [M+H]+: 538

Example 112c (2R,6S)-Diethyl1-benzyl-4-(phenylsulfonyl)piperazine-2,6-dicarboxylate 112c

To a solution of 112b (20g, 38.8 mmol) in toluene (100 ml) was addedBnNH₂ (12.48 g, 116.5 mmol). The reaction mixture was stirred at 90° C.for 3 h. Then the mixture was purified through a silica gel columneluting with PE:EA=50:1 to 10:1 to give 112c as a white crystal (10.7 g,60%) MS: [M+H]+: 461

Example 112d (1-Benzyl-4-(phenylsulfonyl)piperazine-2,6-diyl)dimethanol112d

1 M solution of LiAlH₄ in THF (70 ml; 70 mmol) was added dropwise undercooling and stirring to 112c (10.7 g; 23.2 mmol) in THF (275 ml). Thereaction mixture was refluxed for 20 min, poured on a saturated solutionof Na₂CO₃ and extracted with TBME three times. The combined organicphases were dried over Na₂SO₄ and evaporated to dryness to yield acolorless solid, which was washed with TBME to yield 112d as whitecrystals (7 g, 80%). MS: [M+H]+: 377

Example 112e9-Benzyl-3-(phenylsulfonyl)-7-oxa-3,9-diaza-bicyclo[3.3.1]nonane 112e

SOCl₂ (1.34 ml; 18.5 mmol) in toluene (14 ml) was added under stirringat room temperature rapidly dropwised to a solution of 112e (7.0 g,18.57 mmol) in DMF (276 ml). The reaction mixture was heated in an oilbath (170° C.) under reflux for 5 h. The reaction mixture wasevaporated, taken up in a saturated solution of Na₂CO₃ and extractedwith EtOAc three times. The combined organic phases were dried overNa₂SO₄, filtered and evaporated to dryness and purified through a silicagel column eluting with PE: EA=10:1 to 5:1 to give 112e as a colorlesscrystal (3.0 g, 45%) MS: [M+H]+: 413

Example 112f 3-(Phenylsulfonyl)-7-oxa-3,9-diaza-bicyclo[3.3.1]nonane112f

To a solution of 112e (3.0 g, 8.37 mmol) in EtOH (100 ml) was added Pd/C(0.5 g) in MeOH (30 ml). The reaction mixture was hydrogenated atatmospheric hydrogen at 50° C. for overnight. Then, the catalyst wasfiltered off, washed with ethanol, and the solvent was evaporated togive 112f as a colorless solid (2.0 g, 90%) MS: [M+H]+: 268

Example 112g9-Methyl-3-(phenylsulfonyl)-7-oxa-3,9-diaza-bicyclo[3.3.1]nonane 112g

To a solution of 112f (2.0 g, 7.5 mmol) in MeCN (60 ml) was added HCHO(1.4 ml, 16 mmol) and 5 drops of AcOH at room temperature. Then NaCNBH₃(1 g, 16 mmol) added and the mixture was stirred for 2 h. It was pouredonto water and extracted with EA (100*3). The organic layer was driedover Na₂SO₄, filtered off, evaporated, and purified through a silica gelcolumn eluting with PE:EA=5:1 to 1:1 to give 112g as a colorless oil(1.5 g, 72%) MS: [M+H]+: 283

Example 112h 9-Methyl-7-oxa-3,9-diaza-bicyclo[3.3.1]nonane Hydrochloride112h

To a solution of 112g (1.5 g, 5.3 mmol) in a mixture of toluene (15 ml)and THF (15 ml) was added LiAlH₄ (0.42 g, 10.8 mmol). The reactionmixture was heated at 110° C. overnight. Then it was poured into HCl (2mol/l). The organic layer was partitioned and the water layer wasevaporated to dryness and dissolved in methanol. The resultingsuspension was filtered and the filtrate was evaporated to give 112h(800 mg, 43%) as a colorless oil. MS: [M+H]+: 143

Example 112i9-Methyl-3-(6-nitropyridin-3-yl)-7-oxa-3,9-diaza-bicyclo[3.3.1]nonane112i

To a solution of 112h (800 mg, 5.6 mmol) and 5-bromo-2-nitropyridine(1.37 g, 6.86 mmol) in DMSO (50 ml) was added Cs₂CO₃ (5g) and t-BuNH₄I(cat.). The reaction mixture was stirred at 120° C. overnight. Then itwas cooled to room temperature, and extracted with EtOAc (300 ml). Theorganic layer was washed with water (3×100 ml) and brine (150 ml), driedover Na₂SO4, filtered and concentrated to get crude product, which waspurified through a silica gel column eluting with DCM:MeOH=100:1 to 50:1to give 112i as a yellow solid (850 mg, 72%) MS: [M+H]+: 265

Example 112j5-(9-Methyl-7-oxa-3,9-diaza-bicyclo[3.3.1]nonan-3-yl)pyridin-2-amine112j

To a solution of 112i (800 mg, 3.0 mmol) in THF (80 ml) was added Pd/C(50 mg) in MeOH (20 ml) at room temperature. The reaction mixture washydrogenated at atmospheric hydrogen pressure and stirred at roomtemperature overnight. The mixture was filtered and the filtrate wasevaporated to give 112j as a colorless oil (680 mg, 90%).MS: [M+H]+: 235

Example 112k5-Bromo-1-methyl-3-(5-(9-methyl-7-oxa-3,9-diaza-bicyclo[3.3.1]-nonan-3-yl)pyridin-2-ylamino)pyridin-2(1H)-one112k

To a solution of 112j (500 mg, 2.1 mmol) and3,5-dibromo-1-methylpyridin-2(1H)-one (852 mg, 3.2 mmol) in dioxane (100ml) was added Xantphos (58 mg, 0.1 mmol), Cs₂CO₃ (2.1 g, 6.4 mmol) andPd₂(dba)₃ (110 mg, 0.1 mmol). The reaction mixture was stirred at 105°C. overnight. The mixture was cooled to RT, filtered, concentrated, andthe crude product was purified through a silica gel column eluting withMeOH:DCM=0 to 1:5 to 112k (500 mg, 46.4%).MS: [M+H]+: 421

Example 112l4-Fluoro-2-(1-methyl-5-(5-(9-methyl-7-oxa-3,9-diaza-bicyclo-[3.3.1]nonan-3-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 1121

A mixture of 112l (500 mg, 1.2 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate (636 mg, 1.32 mmol), PdCl₂(dppf) (95 mg, 0.12 mmol), K₃PO₄ (500mg, 2.4 mmol), and NaOAc (195 mg, 2.4 mmol) in MeCN (8 mL) and H₂O (0.8mL) was heated at 110° C. for 3 h. The solvent was evaporated in vacuo.The residue was purified on silica gel column eluting with DCM:MeOH=50:1 to give 1121 (340 mg, yield 41%). MS: [M+H]+: 696

Example 1122-(5-Fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(9-methyl-7-oxa-3,9-diaza-bicyclo[3.3.1]nonan-3-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one112

A mixture of 112l (340 mg, 0.489 mmol) and LiOH hydrate (445 mg, 24.45mmol) in i-PrOH (4 mL), THF (4 mL) and H₂O (4 mL) was stirred at 30° C.for 1 h. The mixture was evaporated in vacuo, and the residue waspurified on prep-HPLC to give 112 as a low yellow solid (105 mg,32.85%). MS: [M+H]⁺ 654. ¹H NMR (500 MHz, CDCl³) δ 8.46 (d, J=2.5 Hz,1H), 7.87 (d, J=3.0 Hz, 1H), 7.69 (s, 1H), 7.42 (d, J=2.0 Hz, 1H),7.23-7.14 (m, 2H), 6.94 (d, J=2.5 Hz, 1H), 6.84 (m, 2H), 4.52 (d, J=11.5Hz, 1H), 4.31 (m, 2H), 4.12 (m, 5H), 3.89 (m, 3H), 3.69 (s, 3H), 3.43(m, 4H), 2.84 (s, 2H), 5.62-2.54 (m, 7H), 1.88 (m, 2H), 1.78 (m, 2H)

Example 113a (3S)-tert-Butyl3-Methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 113a

The procedures described in Example 104 and starting with(3S)-tert-butyl 3-methylpiperazine-1-carboxylate (10.0 g, 50 mmol) and5-bromo-2-nitropyridine (10.5 g, 50 mmol) afforded 113a as a yellowsolid (8.05 g, 50%). See FIG. 13. MS-ESI: [M+H]⁺ 323

Example 113b (3S)-tert-Butyl4-(6-Aminopyridin-3-yl)-3-methylpiperazine-1-carboxylate 113b

The procedures described in Example 104 and starting with 113a (5.8 g,18 mmol) afforded 113b as a brown solid (4.9 g, 93%). See FIG. 13.MS-ESI: [M+H]⁺ 293

Example 113c (3S)-tert-Butyl4-(6-(5-Bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridine-3-yl)-3-methylpiperazine-1-carboxylate 113c

The procedures described in Example 104 and starting with 113b (4.0 g,13.7 mmol) and 3,5-dibromo-1-methylpyridin-2(1H)-one (5.5 g, 20.6 mmol)afforded 113c as a yellow solid (5.4 g, 83%). See FIG. 13. MS-ESI:[M+H]⁺ 478

Example 113d(3S)-5-Bromo-1-methyl-3-(5-(2-methylpiperazin-1-yl)pyridin-2-ylamino)pyridine-2(1H)-one113d

The procedures described in Example 104 and starting with 113c (3.1 g,6.5 mmol) afforded 113d as a yellow solid (2.3 g, 94%). See FIG. 13.MS-ESI: [M+H]⁺ 378.

Example 113e(S)-5-Bromo-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one113e

A mixture of 113d (40.0 g, 106 mmol), oxetan-3-one (11.4 g, 159 mmol),NaBH₃CN (10.0 g, 159 mmol), and zinc chloride (21.3 g, 159 mmol) inmethanol (700 mL) was stirred at 50° C. for 5 hours. The mixture wasadded to water (100 mL) and concentrated under reduced pressure. Theresidue was extracted with dichloromethane (3×200 mL). The combinedorganic layer was concentrated under reduced pressure and the residuewas purified by silica-gel column chromatography eluting with 40:1dichloromethane/methanol to afford 113e (35 g, 73%). See FIG. 13. MS:[M+H]⁺ 434.

Example 113f(3S)-1-Methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)-pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one113f

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 113e (1.0 g, 1.0 eq.,2.3 mmol), Pin₂B₂ (1.46 g, 2.50 eq., 5.75 mmol), Pd₂(dba)₃ (105 mg, 0.05eq., 0.125 mmol), X-Phos (93 mg, 0.1 eq., 0.23 mmol), potassium acetate(676 mg, 3.0 eq., 6.9 mmol), and dioxane (50 mL). After three cycles ofvacuum/argon flush, the mixture was heated at 90° C. for 4 h. It wasthen cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure and the resulting residue was washedwith 3:1 petroleum ether/ethyl acetate (80 mL) to afford 113f as ayellow solid (1.0 g, 90%). See FIG. 13. MS: [M+H]⁺ 482.

Example 113g 4-[5-(Ethoxycarbonyl)-1H-pyrrol-3-yl]-4-oxobutanoic Acid113g

Into a 3000-mL 4-necked round-bottom flask was placed a solution ofoxolane-2,5-dione (100 g, 999.27 mmol, 2.00 equiv) in 1,2-dichloroethane(690 mL) and AlCl₃ (400.5 g, 3.00 mol, 6.00 equiv), followed by theaddition of a solution of ethyl 1H-pyrrole-2-carboxylate (69 g, 495.86mmol, 1.00 equiv) in 1,2-dichloroethane (660 mL) dropwise with stirringat room temperature over 20 min (FIG. 14). The resulting solution wasstirred at room temperature for 3 h and quenched by the addition of 3 kgof water/ice. The solids were collected by filtration, washed with1×1000 mL of water and dried in a vacuum oven to afford 105g (89%) of113g as a white solid.

Example 113h 4-[5-(Ethoxycarbonyl)-1H-pyrrol-3-yl]butanoic Acid 113h

Into a 2000-mL 4-necked round-bottom flask was placed a solution of 113g(105 g, 438.92 mmol, 1.00 equiv) in CF₃COOH (1000 mL), followed by theaddition of triethylsilane (204 g, 1.75 mol, 4.00 equiv) dropwise withstirring at room temperature over 30 min (FIG. 14). The resultingsolution was stirred at room temperature for 8 h, concentrated undervacuum and diluted with 500 mL of water and 500 mL of ethyl acetate. ThepH value of the solution was adjusted to 7 with saturated aqueous sodiumbicarbonate. The resulting solution was extracted with 3×500 mL of ethylacetate. The combined organic layers were dried over anhydrous sodiumsulfate and concentrated under vacuum to afford 30 g (30%) of 113h as alight brown solid.

Example 113i Ethyl 7-Oxo-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 113i

Into a 1000-mL round-bottom flask was placed a solution of 113h (30 g,133.19 mmol, 1.00 equiv) in CF₃COOH (500 mL) and trifluoroacetyl2,2,2-trifluoroacetate (42 g, 199.97 mmol, 1.50 equiv). See FIG. 14. Theresulting solution was stirred at room temperature for 60 min,concentrated under vacuum, diluted with 500 mL of water and 500 mL ofEA, and extracted with 3×500 mL of ethyl acetate. The combined organiclayers were washed with 1×500 mL of saturated aqueous potassiumcarbonate and 1×500 mL of brine, dried over anhydrous sodium sulfate andconcentrated under vacuum to afford 24 g (87%) of 113i as a light brownsolid.

Example 113j Ethyl6,6-Difluoro-7-oxo-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 113j

Into a 2000-mL 4-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed a solution of 113i (24 g, 115.82mmol, 1.00 equiv) in tetrahydrofuran (200 mL), followed by the additionof LiHMDS (406 mL, 3.50 equiv) dropwise with stirring at −78° C. over 30min (FIG. 14). To this was added a solution ofN-(benzenesulfonyl)-S-phenylfluoranesulfonamido (109.5 g, 347.24 mmol,3.00 equiv) in tetrahydrofuran (500 mL) dropwise with stirring at −78°C. over 30 min. The resulting solution was stirred at room temperatureovernight, quenched by the addition of 200 mL of saturated aqueous NH₄Cland extracted with 3×200 mL of ethyl acetate. The combined organiclayers were dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified on a silica gel column eluting withpetroleum ether/ethyl acetate (30:1) to afford 9.5 g (34%) of 113j as ayellow solid.

Example 113k Ethyl6,6-Difluoro-7-hydroxy-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 113k

Into a 250-mL 3-necked round-bottom flask was placed a solution of 113j(18 g, 74.01 mmol, 1.00 equiv) in ethanol (100 mL), followed by theaddition of NaBH₄ (2.8 g, 74.02 mmol, 1.00 equiv) in several batches at0° C. (FIG. 14). The resulting solution was stirred at 5° C. for 10 min,quenched by the addition of 50 mL of saturated aqueous NH₄Cl,concentrated under vacuum and extracted with 3×50 mL of ethyl acetate.The combined organic layers were washed with 1×100 mL of water and 1×100mL of brine, dried over anhydrous sodium sulfate and concentrated undervacuum to afford 18 g (99%) of 113k as a white solid.

Example 1131 Ethyl6,6-Difluoro-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 113l

Into a 500-mL 3-necked round-bottom flask was placed a solution of 113k(18 g, 73.40 mmol, 1.00 equiv) in dichloromethane (200 mL) and CF₃COOH(41.9 g, 367.48 mmol, 5.00 equiv), followed by the addition oftriethylsilane (25.6 g, 220.16 mmol, 3.00 equiv) dropwise with stirringat 0° C. over 20 min (FIG. 14). The resulting solution was stirred atroom temperature for 4 h, adjusted pH to 7 with saturated aqueous sodiumbicarbonate and extracted with 1×200 mL of dichloromethane. The combinedorganic layers were washed with 1×100 mL of water and 1×100 mL of brine,dried over anhydrous sodium sulfate and concentrated under vacuum. Thecrude product was purified by Flash-Prep-HPLC to afford 10 g (59%) of1131 as a white solid.

Example 113m Ethyl1-(Cyanomethyl)-6,6-difluoro-4,5,6,7-tetrahydro-1H-indole-2-carboxylate113m

Into a 250-mL 3-necked round-bottom flask was placed a solution of 1131(5.0 g, 21.81 mmol, 1.00 equiv) in N,N-dimethylformamide (50 mL),followed by the addition of sodium hydride (1.3 g, 54.17 mmol, 1.40equiv, 60%) in several batches at 0° C. over 10 min. (FIG. 14). To thiswas added 2-bromoacetonitrile (3.7 g, 30.85 mmol, 1.40 equiv) dropwisewith stirring at 20° C. over 10 min. The resulting solution was stirredat room temperature for 7 h, diluted with 100 mL of water and extractedwith 3×50 mL of ethyl acetate. The combined organic layers were washedwith 1×100 mL of water and 1×100 mL of brine, dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluting with ethyl acetate/petroleum ether (1:20)to afford 3.3 g (56%) of 113m as a light yellow oil.

Example 113n Ethyl1-(2-Aminoethyl)-6,6-difluoro-4,5,6,7-tetrahydro-1H-indole-2-carboxylate113n

Into a 100-mL round-bottom flask was placed a solution of 113m (3.3 g,12.30 mmol, 1.00 equiv) in ethanol (30 mL) and NiCl₂.6H₂O (3.2 g, 13.45mmol, 1.10 equiv), followed by the addition of NaBH₄ (1.4 g, 37.01 mmol,3.00 equiv) in several batches at 0° C. (FIG. 14). The resultingsolution was stirred at room temperature for 24 h. The solids werefiltered out and the filtrate was concentrated under vacuum. Theresulting solution was diluted with 30 mL of ethyl acetate and washedwith 1×30 mL of hydrogen chloride (2/V). The solution was adjusted topH=9 with saturated aqueous sodium bicarbonate and extracted with 2×30mL of ethyl acetate. The combined organic layers were dried overanhydrous sodium sulfate and concentrated under vacuum to afford 0.7 g(21%) of 113n as a light yellow oil.

Example 113o7,7-difluoro-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-1-one 113o

Into a 250-mL round-bottom flask was placed a solution of 113n (10 g,36.73 mmol, 1.00 equiv) in toluene (100 mL) and acetic acid (1.1 g,18.32 mmol, 0.50 equiv). See FIG. 14. The resulting solution was heatedto reflux for 2 h, cooled and concentrated under vacuum. The residue wastriturated in 100 mL of dry ether. The crude product was purified byrecrystallization from ethanol to afford 4.43 g (53%) of 113o as a whitesolid. MS-ESI: [M+H]⁺ 227. ¹H NMR (300 MHz, DMSO) δ 2.10-2.24 (2H, m),2.59-2.64 (2H, m), 3.17-3.27 (2H, m), 3.43-3.48 (2H, m), 3.88-3.92 (2H,m), 6.44 (1H, s), 7.56 (1H, s).

Example 113p2-Bromo-6-(7,7-difluoro-1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-4-fluorobenzylAcetate 113p

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with 1,4-dioxane (40 mL), 113o (890 mg, 3.94 mmol),2,6-dibromo-4-fluorobenzyl acetate 101c (3847 mg, 11.8 mmol), Pd₂(dba)₃(180 mg, 0.197 mmol), XantPhos (227 mg, 0.394 mmol), and cesiumcarbonate (2.57 g, 7.88 mmol). After three cycles of vacuum/argon flush,the mixture was heated at 100° C. for 16 h. It was then filtered and thefiltrate was evaporated under reduced pressure. The residue was purifiedby silica-gel column chromatography eluting with 3:1 petroleumether/ethyl acetate to afford 113p (1100 mg, 62%) as a white solid.MS-ESI: [M+H]⁺ 471.1.

Example 113q(S)-2-(7,7-Difluoro-1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-4-fluoro-6-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)benzylAcetate 113q

A sealed tube was charged with 113p (47 mg, 0.10 mmol), 113f (47 mg,0.10 mmol), Pd(dppf)Cl₂ (4 mg, 0.005 mmol), sodium acetate (16 mg, 0.2mmol,), K₃PO₄ (43 mg, 0.2 mmol), acetonitrile (2 mL), and water (0.2mL). After three cycles of vacuum/argon flush, the mixture was heated at100° C. for 1 h. It was then filtered and the filtrate was evaporatedunder reduced pressure. The residue was purified by silica-gel columnchromatography eluting with 25:1 dichloromethane/methanol to afford 113q(37 mg, 50%) as a brown solid. MS: [M+H]⁺ 746.3

Example 113(S)-7,7-difluoro-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one113

A mixture of 113q (37 mg, 0.05 mmol) and lithium hydroxide (12 mg, 0.5mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at 30°C. for 1 h. The mixture was evaporated under reduced pressure. Water (10mL) was added to the residue and the resulting mixture was extractedwith ethyl acetate (2×10 mL). The combined organic layer wasconcentrated under reduced pressure and the residue was purified byreverse-phase prep-HPLC to afford 113 (20 mg, 57%) as a white solid. MS:[M+H]⁺ 704.3. ¹H NMR (500 MHz, CDCl₃) δ 8.57-8.54 (m, 1H), 7.95 (d,J=3.0 Hz, 1H), 7.82 (d, J=3.5 Hz, 1H), 7.45-7.41 (m, 1H), 7.32 (d, J=8.5Hz, 1H), 7.19-7.16 (m, 1H), 6.99-6.95 (m, 1H), 6.88 (d, J=6.5 Hz, 1H),6.82 (d, J=8.5 Hz, 1H), 5.97 (d, J=11.5 Hz, 1H), 4.71-4.64 (m, 4H), 4.54(d, J=12.0 Hz, 1H), 4.35-4.18 (m, 5H), 3.99-3.95 (m, 1H), 3.71 (s, 3H),3.53 (t, J=6.0 Hz, 1H), 3.47-3.45 (m, 1H), 3.17 (t, J=12.0 Hz, 1H), 3.08(t, J=5.0 Hz, 2H), 2.90 (t, J=8.5 Hz, 1H), 2.79 (t, J=6.0 Hz, 1H),2.66-2.63 (m, 1H), 2.56 (d, J=11.0 Hz, 1H), 2.49-2.47 (m, 2H), 2.30-2.20(m, 2H), 0.99 (d, J=6.0 Hz, 3H).

Example 114a (S)-tert-Butyl3-Ethyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 114a

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (50 mL),5-bromo-2-nitropyridine (2.02 g, 10 mmol), (S)-tert-butyl3-ethylpiperazine-1-carboxylate (2.14 g, 10.0 mmol), Pd₂(dba)₃ (458 mg,0.50 mmol), XantPhos (576 mg, 1.0 mmol), and cesium carbonate (6.52 g,20 mmol). After three cycles of vacuum/argon flush, the mixture washeated at 100° C. overnight. After this time the reaction was cooled toroom temperature. It was then filtered and the filtrate was evaporatedunder reduced pressure. The residue was purified by silica-gel columnchromatography eluting with 3:1 petroleum ether/ethyl acetate to afford114a (700 mg, 22%) as a yellow solid. MS: [M+H]⁺ 336

Example 114b (S)-tert-Butyl4-(6-Aminopyridin-3-yl)-3-ethylpiperazine-1-carboxylate 114b

A 100-mL single-neck round-bottomed flask was purged with nitrogen andcharged with 114a (0.7 g, 2.08 mmol), 10% palladium on carbon (50% wet,208 mg), and methanol (40 mL). The mixture was evacuated, charged withhydrogen gas, and stirred at room temperature for 6 h. The hydrogen wasthen evacuated and nitrogen was charged into the flask. The catalyst wasremoved by filtration through a pad of Celite and the filtrate wasconcentrated under reduced pressure to afford 114b (568 mg, 89%). MS:[M+H]⁺ 306

Example 114c (S)-tert-Butyl4-(6-(5-Bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)-3-ethylpiperazine-1-carboxylate 114c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (50 mL),114b (568 mg, 1.86 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (498 mg,1.86 mmol), Pd₂(dba)₃ (85 mg, 0.093 mmol), XantPhos (107 mg, 0.186mmol), and cesium carbonate (1.198 g, 3.72 mmol). After three cycles ofvacuum/argon flush, the mixture was heated at 100° C. for 6 h. It wasthen filtered and the filtrate was evaporated under reduced pressure.The residue was purified by silica-gel column chromatography elutingwith 100:1 dichloromethane/methanol to afford 114c (502 mg, 55%) as ayellow solid. MS: [M+H]⁺ 492.

Example 114d(S)-5-Bromo-3-(5-(2-ethylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one114d

A mixture of 114c (502 mg, 1.02 mmol), dichloromethane (2 mL), and 4.0 MHCl/dioxane (4 mL) was stirred at room temperature for 5 h. It was thenconcentrated under reduced pressure to afford crude 114d as a yellowsolid (263 mg, 66%), which was used in the next step without furtherpurification. MS: [M+H]⁺ 392.

Example 114e(S)-5-Bromo-3-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one114e

A mixture of 114d (263 mg, 0.67 mmol), oxetan-3-one (96 mg, 1.34 mmol),NaBH₃CN 104 mg, 1.68 mmol), and zinc chloride (227 mg, 1.68 mmol) inmethanol (10 mL) was stirred at 50° C. for 5 hours. water (10 mL) wasthen added to the reaction. The resulting mixture was concentrated underreduced pressure. The residue was extracted with dichloromethane threetimes. The combined organic layer was concentrated under reducedpressure and the residue was purified by silica-gel columnchromatography eluting with 50:1 dichloromethane/methanol to afford 114e(203 mg, 68%). MS: [M+H]⁺ 448.

Example 114f(S)-3-(5-(2-Ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one114f

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 114e (3219 mg, 7.20mmol), Pin₂B₂ (9072 mg, 36.0 mmol), Pd₂(dba)₃ (329 mg, 0.36 mmol),X-phos (302 mg, 0.72 mmol), potassium acetate (2117 mg, 21.6 mmol), anddioxane (50 mL). After three cycles of vacuum/argon flush, the mixturewas heated at 60° C. for 16 h. It was then cooled to room temperatureand filtered. The filtrate was concentrated under reduced pressure andthe resulting residue was washed with 8:1 petroleum ether/ethyl acetate(80 mL) to afford 114f as a yellow solid (3.0 g, 84%). MS: [M+H]⁺ 496.4.

Example 114g(2-{4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-6-[5-({5-[(2S)-2-ethyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-4-fluorophenyl)methylAcetate 114g

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 114f (180 mg, 0.40mmol),(2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 103g (198 mg, 0.40 mmol), Pd(dppf)Cl₂ (29 mg, 0.04 mmol), K₃PO₄(170 mg, 0.8 mmol), sodium acetate (66 mg, 0.8 mmol), acetonitrile (5mL), and water (1.0 mL). After three cycles of vacuum/argon flush, themixture was heated at reflux for 2 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 114gas a yellow solid (115 mg, 39%). MS: [M+H]⁺ 738.4

Example 1142-(3-{5-[5-((S)-2-Ethyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridin-3-yl}-5-fluoro-2-hydroxymethyl-phenyl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H,6H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1-one114

A mixture of 114g (115 mg, 0.16 mmol) and lithium hydroxide (38 mg, 1.6mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at 30°C. for 1 h. The mixture was evaporated under reduced pressure and theresidue was extracted with ethyl acetate (2×10 mL). The combined ethylacetate extract was concentrated under reduced pressure and the residuewas purified by reverse-phase prep-HPLC to afford 114 (45 mg, 40%) as awhite solid. MS: [M+H]⁺ 696.4. ¹H NMR (500 MHz, CDCl₃) δ 8.55-8.54 (m,1H), 7.91 (s, 1H), 7.80 (d, J=1.5 Hz, 1H), 7.47 (s, 1H), 7.26 (d, J=3.0Hz, 1H), 7.17 (d, J=9.0 Hz, 1H), 6.95 (dd, J=2.5, 8.5 Hz, 1H), 6.83-6.81(m, 2H), 4.71 (t, J=6.5 Hz, 2H), 4.67 (t, J=6.0 Hz, 1H), 4.62 (t, J=6.0Hz, 1H), 4.57-4.55 (m, 1H), 4.41-4.38 (m, 1H), 4.32-4.30 (m, 1H),4.24-4.14 (m, 3H), 3.92-3.87 (m, 1H), 3.71 (s, 3H), 3.56-3.50 (m, 1H),3.33-3.29 (m, 1H), 3.13-3.11 (m, 2H), 2.58-2.56 (m, 3H), 2.52 (s, 2H),2.44-2.43 (m, 2H), 2.38-2.32 (m, 1H), 1.66-1.64 (m, 1H), 1.42-1.37 (m,1H), 1.28 (s, 6H), 0.82 (t, J=7.0 Hz, 3H).

Example 115a (R)-tert-Butyl3-Methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 115a

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (60 mL),5-bromo-2-nitropyridine (2.0 g, 10.0 mmol), (R)-tert-butyl3-methylpiperazine-1-carboxylate (2.0 g, 10.0 mmol), and cesiumcarbonate (6.5 g, 20 mmol). After bubbling nitrogen through theresulting mixture for 10 minutes,tris(dibenzylideneacetone)dipalladium(0) (915 mg, 1.0 mmol) and XantPhos(579 mg, 1.0 mmol) were added. The system was subjected to three cyclesof vacuum/argon flush and heated at 100° C. for 15 h. After this timethe reaction was cooled to room temperature and filtered. The filtratewas partitioned between ethyl acetate (100 mL) and water (100 mL). Theaqueous layer was separated and extracted with ethyl acetate (3×50 mL).The combined organic layer was washed with brine (100 mL) and dried oversodium sulfate. The drying agent was removed by filtration and thefiltrate was concentrated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with 30:1dichloromethane/methanol to afford 115a (1.6 g, 44%) as a yellow solid.MS-ESI: [M+H]⁺ 323. ¹H NMR (500 MHz, DMSO-d₆) δ 8.21 (d, J=3.5 Hz, 1H),8.18 (d, J=9.0 Hz, 1H), 7.45-7.43 (m, 1H), 4.34-4.33 (m, 1H), 3.92-3.99(m, 1H), 3.80 (d, J=12.5 Hz, 2H), 3.06-3.23 (m, 3H), 1.43 (s, 9H), 1.09(d, J=6.5 Hz, 3H).

Example 115b (R)-tert-Butyl4-(6-Aminopyridin-3-yl)-3-methylpiperazine-1-carboxylate 115b

A 250-mL flask was purged with nitrogen and charged with 115a (1.5 g,4.6 mmol), 10% palladium on carbon (50% wet, 200 mg), and methanol (70mL). It was evacuated, charged with hydrogen gas, and stirred at roomtemperature for 10 h. The hydrogen was then evacuated and nitrogen wascharged into the flask. The catalyst was removed by filtration through apad of Celite and the filtrate was concentrated under reduced pressureto afford 115b (1.1 g, 81%) as a brown solid. MS-ESI: [M+H]⁺ 293

Example 115c (R)-tert-Butyl4-(6-(5-Bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)-3-methylpiperazine-1-carboxylate115c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (40 mL),115b (1.0 g, 3.4 mmol), 3,5-dibromo-1-methylpyridin-2(1H)-one (2.7 g,10.2 mmol), and cesium carbonate (2.2 g, 6.8 mmol). After bubblingnitrogen through the resulting mixture for 10 minutes, XantPhos (198 mg,0.34 mmol) and tris(dibenzylideneacetone)dipalladium(0) (313 mg, 0.34mmol) were added. The reaction mixture was subjected to three cycles ofvacuum/argon flush and heated at 100° C. for 5 h. After this time thereaction was cooled to room temperature and filtered. The filtrate waspartitioned between ethyl acetate (50 mL) and water (50 mL). The aqueouslayer was separated and extracted with ethyl acetate (3×30 mL). Thecombined organic layer was washed with brine (50 mL) and dried oversodium sulfate. The drying agent was removed by filtration and thefiltrate was concentrated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with 30:1dichloromethane/methanol to afford 115c as a yellow solid (1.1 g, 63%).MS-ESI: [M+H]⁺ 478.

Example 115d(R)-5-Bromo-1-methyl-3-(5-(2-methylpiperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one115d

To a mixture of 115c (600 mg, 1.26 mmol) in methanol (20 mL) was addedHCl/dioxane (4M, 4 mL). The reaction mixture was stirred at roomtemperature for 4 h. It was then concentrated under reduced pressure.The residue was basified with aqueous 1M NaOH and extracted withdichloromethane (3×30 mL). The combined organic layer was washed withbrine and concentrated under reduced pressure to afford 115d (450 mg,95%) as a yellow solid. MS-ESI: [M+H]⁺ 378.

Example 115e(R)-5-Bromo-3-(5-(2,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one115e

A mixture of 115d (500 mg, 1.3 mmol) and 30% formaldehyde (650 mg, 6.5mmol) in methanol/acetic acid (30 mL/3 mL) was stirred at roomtemperature for 5 minutes, followed by the addition of NaBH₃CN (120 mg,1.9 mmol). The mixture was stirred at room temperature for 4 h. water(20 mL) was added and the resulting mixture was concentrated underreduced pressure. The residue was extracted with dichloromethane (3×30mL). The combined organic layer was concentrated under reduced pressureand the residue was purified by silica-gel column chromatography elutingwith 30:1 dichloromethane/methanol to afford 115e as a yellow solid (473mg, 92%). MS-ESI: [M+H]⁺ 392.

Example 115f(R)-5-Bromo-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one115f

A mixture of 115e (40.0 g, 106 mmol), oxetan-3-one (11.4 g, 159 mmol),NaBH₃CN (10.0 g, 159 mmol), and zinc chloride (21.3 g, 159 mmol) inmethanol (700 mL) was stirred at 50° C. for 5 hours. water (50 mL) wasadded to the mixture and concentrated under reduced pressure. Theresidue was extracted with dichloromethane (3×200 mL) and the combinedorganic layer was concentrated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with 40:1dichloromethane/methanol to afford 115f (35 g, 73%). MS: [M+H]⁺ 434.

Example 115g2,2,2-Trichloro-1-(4,5,6,7-tetrahydro-1H-indol-2-yl)ethanone 115g

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer, condenser and nitrogen inlet was purged with nitrogen andcharged with 4,5,6,7-tetrahydro-1H-indole (3.00 g, 24.8 mmol),trichloroacetyl chloride (13.5 g, 74.4 mmol) and 1,2-dichloroethane (50mL). The solution was stirred at 85° C. for 2 h. After that time, thereaction mixture was concentrated under reduced pressure to afford a100% yield (6.50 g) of 115g as a black semi-solid: ¹H NMR (500 MHz,DMSO-d₆) δ 11.94 (s, 1H), 7.05 (s, 1H), 2.62 (t, 2H, J=6.0 Hz), 2.47 (t,2H, J=6.0 Hz), 1.80 (m, 2H), 1.65 (m, 2H); MS (ESI+) m/z 266.0 (M+H)

Example 115h Ethyl 4,5,6,7-Tetrahydro-1H-indole-2-carboxylate 115h

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged with115g (6.50 g, 24.8 mmol), sodium ethoxide (17.0 mg, 0.25 mmol) andethanol (40 mL). The solution was stirred at room temperature for 1 h.After that time, the reaction mixture was concentrated under reducedpressure. The residue was purified by column chromatography to afford a100% yield (4.80 g) of 115h as a brown solid: mp 70-72° C.; ¹H NMR (300MHz, CDCl₃) δ 9.08 (s, 1H), 6.75 (s, 1H), 4.25 (q, 2H, J=7.2 Hz), 2.65(t, 2H, J=6.0 Hz), 2.56 (t, 2H, J=6.0 Hz), 1.85 (m, 4H), 1.28 (t, 3H,J=7.2 Hz); MS (ESI+) m/z 194.1 (M+H)

Example 115i Ethyl1-(Cyanomethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 115i

A 125-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged with115h (5.76 g, 29.8 mmol) and DMF (50 mL). The solution was cooled to 0°C. using an ice bath. NaH (60% dispersion in mineral oil, 1.43 g, 35.8mmol) was added. The resulting mixture was stirred at room temperaturefor 1 h. After that time, bromoacetonitrile (1.43 g, 35.8 mmol) wasadded. The mixture was stirred at room temperature for 14 h. After thattime, the reaction mixture was concentrated under reduced pressure andthe residue was partitioned between ethyl acetate (150 mL) and water(450 mL). The organic layer was separated, and the aqueous layer wasextracted with ethyl acetate (3×150 mL). The combined organic layerswere washed with brine, dried over sodium sulfate and concentrated underreduced pressure. The residue was purified by column chromatography toafford a 55% yield (3.80 g) of 115i as a yellow semi-solid: ¹H NMR (300MHz, CDCl₃) δ 6.66 (s, 1H), 5.29 (s, 2H), 4.28 (q, 2H, J=7.2 Hz), 2.62(t, 2H, J=6.3 Hz), 2.49 (t, 2H, J=6.3 Hz), 1.92 (m, 2H), 1.75 (m, 2H),1.33 (t, 3H, J=7.2 Hz); MS (ESI+) m/z 233.1 (M+H)

Example 115j Ethyl1-(2-Aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 115j

A 200-mL Parr reactor bottle was purged with nitrogen and charged with10% palladium on carbon (50% wet, 1.28 g dry weight), 115i (3.00 g, 12.9mmol), 12% hydrochloric acid (6.5 mL, 25 mmol), ethyl acetate (60 mL)and ethanol (40 mL). The bottle was attached to a Parr hydrogenator,evacuated, charged with hydrogen gas to a pressure of 50 psi and shakenfor 6 h. After this time, the hydrogen was evacuated, and nitrogen wascharged into the bottle. Celite 521 (4.0 g) was added, and the mixturewas filtered through a pad of Celite 521. The filter cake was washedwith ethanol (2×20 mL), and the combined filtrates were concentrated todryness under reduced pressure. The residue was partitioned betweenethyl acetate (150 mL) and 10% aqueous potassium carbonate (100 mL). Theorganic layer was separated, and the aqueous layer was extracted withethyl acetate (3×75 mL). The combined organic layers were dried oversodium sulfate and concentrated under reduced pressure. The residue wastriturated with ethanol (5 mL) to afford a 71% yield (1.71 g) of 115j asa white solid: mp 102-104° C.; ¹H NMR (500 MHz, DMSO-d₆) δ 6.61 (s, 1H),6.22 (br, 2H), 4.15 (m, 4H), 2.77 (m, 2H), 2.59 (t, 2H, J=6.5 Hz), 2.42(t, 2H, J=6.5 Hz), 1.70 (m, 2H), 1.62 (m, 2H), 1.23 (t, 3H, J=7.0 Hz);MS (APCI+) m/z 237.2 (M+H)

Example 115k 3,4,6,7,8,9-Hexahydropyrazino[1,2-a]indol-1(2H)-one 115k

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged withethyl 1-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 115j(1.80 g, 7.63 mmol), sodium ethoxide (1.55 g, 22.8 mmol) and ethanol (50mL). The mixture was stirred at 55° C. for 5 h. After that time, thereaction mixture was concentrated under reduced pressure and the residuewas partitioned between ethyl acetate (200 mL) and water (100 mL). Theorganic layer was separated, and the aqueous layer was extracted withethyl acetate (2×100 mL). The combined organic layers were washed withbrine, dried over sodium sulfate and concentrated under reducedpressure. The residue was purified by column chromatography to afford a42% yield (605 mg) of 115k as a white solid: mp 207-209° C.; ¹H NMR (500MHz, DMSO-d₆) δ 7.41 (s, 1H), 6.36 (s, 1H), 3.84 (t, 2H, J=6.0 Hz), 3.42(m, 2H), 2.51 (t, 2H, J=6.0 Hz), 2.42 (t, 2H, J=6.0 Hz), 1.76 (m, 2H),1.65 (m, 2H); (APCI+) m/z 191.3 (M+H)

Example 115l2-Bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylAcetate 115l

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 115k (3.8 g, 20 mmol),2,6-dibromo-4-fluorobenzyl acetate 101c (20.0 g, 61 mmol), XantPhos(1.16 g, 2.0 mmol), tris(dibenzylideneacetone)dipalladium(0) (1.83 g,2.0 mmol), Cs₂CO₃ (16.3 g, 50 mmol), and 1,4-dioxane (120 mL). Thesystem was evacuated and then refilled with N₂. A reflux condenser wasattached to the flask and the reaction mixture was heated at 100° C. for16 h. The mixture was cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure and the resultingresidue was purified by silica-gel column chromatography eluting with5:1 petroleum ether/ethyl acetate to afford 115l as a white solid (5.2g, 60%). MS: [M+H]⁺ 435. ¹H NMR (500 MHz, DMSO-d₆) δ 7.71-7.69 (m, 1H),7.49-7.47 (m, 1H), 6.52 (s, 1H), 5.01 (m, 2H), 4.18 (m, 2H), 4.02 (m,1H), 3.73 (m, 1H), 2.60 (m, 2H), 2.45 (m, 2H), 1.98 (s, 3H), 1.77 (m,2H), 1.68 (m, 2H).

Example 115m4-Fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylAcetate 115m

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 115l (3.8 g, 8.8 mmol), (PinB)₂ (10.9 g, 43mmol), Pd(dppf)Cl₂ (0.37 g, 0.50 mmol), potassium acetate (2.55 g, 26mmol), and 1,4-dioxane (150 mL). The system was evacuated and thenrefilled with N₂. A reflux condenser was attached to the flask and thereaction mixture was heated at 100° C. for 15 h. The mixture was cooledto room temperature and filtered. The filtrate was concentrated underreduced pressure and the resulting residue was purified by silica-gelcolumn chromatography eluting with 5:1 petroleum ether/ethyl acetate toafford 115m as a yellow solid (3.2 g, 75%). MS: [M+H]⁺ 483.

Example 115n(R)-4-Fluoro-2-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylAcetate 115n

Following the procedure in Example 102 and starting with 115e (220 mg,0.50 mmol, 1.0 eq.), 115m (482 mg, 1.0 mmol, 2.0 eq.) afforded 115n as ayellow solid (195 mg, 55%). MS: [M+H]+ 710.4

Example 115(R)-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one115

Following the procedure in Example 102 and starting with 115n (190 mg,0.27 mmol) afforded 115 as a white solid (47 mg, 26%). MS: [M+H]+ 668.4.¹H NMR (500 MHz, DMSO-d₆) δ 8.58 (s, 1H), 8.42 (s, 1H), 7.84 (d, J=3.0Hz, 1H), 7.36-7.32 (m, 3H), 7.25-7.18 (m, 2H), 6.52 (s, 1H), 4.88 (s,1H), 4.56-4.42 (m, 4H), 4.31-4.30 (m, 2H), 4.18-4.13 (m, 3H), 3.89-3.88(m, 1H), 3.68-3.67 (m, 1H), 3.58 (s, 3H), 3.39-3.38 (m, 1H), 3.08-3.07(m, 1H), 2.94-2.93 (m, 1H), 2.51-2.45 (m, 5H), 2.33-2.32 (m, 2H),2.19-2.18 (m, 1H), 1.79-1.69 (m, 4H), 0.93-0.92 (m, 3H).

Example 116a{2-[5-({5-[(2S,5R)-2,5-Dimethyl-4-(oxetan-3-yl)piperazin-1-yl]pyridine-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-6-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,5)]dodeca-2(6),7-dien-10-yl}-4-fluorophenyl}methylAcetate 116a

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with5-bromo-3-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one104e (134 mg, 0.30 mmol),1-methyl-3-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetra-methyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one103g (298 mg, 0.6 mmol), Pd(dppf)Cl₂ (22 mg, 0.03 mmol), K₃PO₄ (127 mg,0.6 mmol), sodium acetate (49 mg, 0.6 mmol), acetonitrile (5 mL), andwater (1.0 mL). After three cycles of vacuum/argon flush, the mixturewas heated at reflux for 2 h. It was then cooled to room temperature andfiltered. The filtrate was concentrated under reduced pressure and theresulting residue was purified by silica-gel column chromatographyeluting with 30:1 dichloromethane/methanol to afford 116a as yellowsolid (150 mg, 68%). MS: [M+H]⁺ 738.3

Example 1162-(3-{5-[5-((2S,5R)-2,5-Dimethyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridin-3-yl}-5-fluoro-2-hydroxymethyl-phenyl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H,6H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1-one116

A mixture of 116a (150 mg, 0.20 mmol) and lithium hydroxide (48 mg, 2.0mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at 30°C. for 1 h. The mixture was evaporated under reduced pressure. Theresulting residue was extracted with ethyl acetate (2×10 mL). Thecombined organic layer was concentrated under reduced pressure and theresidue was purified by reverse-phase prep-HPLC to afford 116 (57 mg,41%) as white solid. MS: [M+H]⁺ 696.3. ¹H NMR (500 MHz, CDCl₃) δ 8.60(dd, J=2.0, 6.5 Hz, 1H), 8.02 (d, J=2.5 Hz, 1H), 7.88 (s, 1H), 7.50-7.49(m, 1H), 7.37 (d, J=8.5 Hz, 1H), 7.16 (d, J=9.0 Hz, 1H), 6.96 (dd,J=2.5, 9.0 Hz, 1H), 6.83-6.81 (m, 2H), 4.77-4.74 (m, 2H), 4.66-4.62 (m,2H), 4.57-4.55 (m, 1H), 4.33-4.31 (m, 1H), 4.23-4.14 (m, 3H), 3.92-3.89(m, 1H), 3.78-3.76 (m, 1H), 3.71 (s, 3H), 3.22-3.20 (m, 1H), 2.93-2.91(m, 1H), 2.75-2.73 (m, 2H), 2.58 (s, 2H), 2.52 (s, 3H), 1.97-1.90 (m,2H), 1.28 (s, 6H), 0.91 (d, J=5.5 Hz, 6H).

Example 117aN-tert-Butyl-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide 117a

A mixture of 4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxylic acid (500g, 2.75 mol, 1.0 equiv) and thionyl chloride (655 g, 5.5 mol, 2.0 equiv)was boiled under reflux for 3 h. Excess thionyl chloride was removed bydistillation under reduced pressure. The residue was taken up indichloromethane (1.0 L) and a solution of tert-butylamine (402 g, 5.5mol, 2.0 equiv) in dichloromethane (500 mL) was added with stirringwhile the temperature of the mixture was kept below 10° C. The resultingsolution was stirred at 25° C. for 16 h. Most of the solvent was removedunder reduced pressure. The residue was chilled in an ice-bath and 2MKOH solution was introduced slowly to adjust the pH to 11 with stirring.The suspension was filtered and the solid was collected, washed threetimes with water, and dried in vacuo to afford 117a as a white solid(580 g, 80%, over two steps). MS: [M+H]⁺ 238. ¹H NMR (500 MHz, CDCl₃) δ7.02 (s, 1H), 5.77 (s, 1H), 2.65 (t, J=6.0 Hz, 1H), 2.47 (t, J=6.0 Hz,1H), 1.74-1.70 (m, 4H), 1.35 (s, 9H).

Example 117bN-tert-Butyl-3-(diazenylmethyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide117b

To a solution of 117a (100 g, 0.42 mol, 1.0 equiv) in THF (500 mL) wasslowly to added n-BuLi (672 mL, 2.5M in THF, 1.68 mol, 4.0 equiv) at−78° C. under argon. The mixture was stirred for 2 h. DMF (306 g, 4.2mol, 10.0 equiv) was added to the mixture while the temperature wasmaintained at −78° C. After another 2.0 h, the reaction was quenchedwith methanol (500 mL) at −78° C. It was stirred for 0.50 h at roomtemperature. 80% aqueous hydrate hydrazine (131 g, 2.1 mol) was addedand the mixture was refluxed at 65° C. overnight. The organic solventwas removed under reduced pressure. The residue was filtered and theyellow solid collected was washed with water. The solid was dried invacuo to afford crude 117b, which was used in the next step withoutfurther purification. MS: [M+H]⁺ 280.

Example 117c8-Thia-4,5-diazatricyclo[7.4.0.0^(2,7)]-trideca-(9),2(7),3-trien-6-one117c

A mixture of 117b (40 g, 144 mmol) in H₂SO₄ (30% aqueous, 3 L) wasrefluxed at 105° C. for 24 h. It was then filtered and the filtrate wasextracted by dichloromethane (3×1 L). The combined extract was driedover Na₂SO₄ and evaporated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with 100:1dichloromethane/methanol to afford 117c as a white solid (9.0 g, 31%).MS: [M+H]⁺ 207. ¹H NMR (500 MHz, CDCl₃) δ 8.15 (s, 1H), 2.96-2.94 (m,2H), 2.86-2.84 (m, 2H), 1.96-1.94 (m, 4H).

Example 117d(2-Bromo-4-fluoro-6-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-5-yl}phenyl)methylacetate 117d

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 117c (1.0 g, 4.85 mmol),2,6-dibromo-4-fluorobenzyl acetate 101c (4.8 g, 14.6 mmol), copper(I)iodide (553 mg, 2.9 mmol), N¹,N²-dimethylethane-1,2-diamine (512 mg,5.82 mmol), Cs₂CO₃ (3.2 g, 9.7 mmol), and 1,4-dioxane (50 mL). Thesystem was evacuated and then refilled with N₂. A reflux condenser wasattached to the flask, and the reaction mixture was heated at 100° C.for 16 h. It was then cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure and the resultingresidue was purified by silica-gel column chromatography eluting with5:1 petroleum ether/ethyl acetate to afford 117d as a yellow solid (437mg, 20%). MS: [M+H]⁺ 451.

Example 117e(4-Fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylAcetate 117e

Following the procedure in Example 104 and starting with 117d (400 mg0.88 mmol) afforded 117e as a yellow solid (353 mg, 80%). MS: [M+H]⁺ 499

Example 117f{4-Fluoro-2-[1-methyl-5-({5-[(2R)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxopyridin-3-yl]-6-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-5-yl}phenyl}methylAcetate 117f

Following the procedure in Example 102 and starting with(R)-5-bromo-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one115f (217 mg, 0.50 mmol) and 117e (249 mg, 0.50 mmol) afforded 117f as ayellow solid (174 mg, 48%). MS: [M+H]⁺ 726.

Example 1173-(5-Fluoro-2-hydroxymethyl-3-{1-methyl-5-[5-((R)-2-methyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-6,7,8,9-tetrahydro-3H-benzo[4,5]thieno[2,3-d]pyridazin-4-one117

Following the procedure in Example 102 and starting with 117f (72 mg,0.10 mmol) afforded 117 as a yellow solid (35 mg, 51%). LCMS: [M+H]⁺684. ¹H NMR (500 MHz, DMSO-d₆) δ 8.56 (d, J=2.0 Hz, 1H), 8.48 (s, 1H),8.43 (s, 1H), 7.85 (d, J=2.5 Hz, 1H), 7.37-7.32 (m, 4H), 7.24 (d, J=9.0Hz, 1H), 4.60 (t, J=5.5 Hz, 1H), 4.57-4.53 (m, 2H), 4.47-4.41 (m, 2H),4.28-4.27 (d, J=4.0 Hz, 2H), 3.68-3.66 (m, 1H), 3.58 (s, 3H), 3.40-3.38(m, 1H), 3.10-3.07 (m, 1H), 2.93-2.91 (m, 3H), 2.84-2.82 (m, 2H),2.54-2.52 (m, 1H), 2.32-2.30 (m, 2H), 2.19-2.18 (m, 1H), 1.89-1.84 (m,4H), 0.93 (t, J=6.5 Hz, 2H).

Example 118a{2-[5-({5-[(2S,5R)-2,5-Dimethyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-4-fluoro-6-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-5-yl}phenyl}methylAcetate 118a

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with5-bromo-3-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one104e (179 mg, 0.40 mmol),(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]-trideca-1(9),2(7),3-trien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 117d (200 mg, 0.40 mmol), Pd(dppf)Cl₂ (29 mg, 0.04 mmol), K₃PO₄(170 mg, 0.8 mmol), sodium acetate (66 mg, 0.8 mmol), acetonitrile (5mL), water (1.0 mL). After three cycles of vacuum/argon flush, themixture was heated at reflux for 2 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 118aas yellow solid (100 mg, 34%). MS: [M+H]⁺ 740.3

Example 1183-(3-{5-[5-((2S,5R)-2,5-Dimethyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridin-3-yl}-5-fluoro-2-hydroxymethyl-phenyl)-6,7,8,9-tetrahydro-3H-benzo[4,5]thieno[2,3-d]pyridazin-4-one118

A mixture of 118a (100 mg, 0.135 mmol) and lithium hydroxide (33 mg,1.35 mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at30° C. for 1 h. The mixture was evaporated under reduced pressure. Theresulting residue was extracted with ethyl acetate (2×10 mL). Thecombined organic layer was concentrated under reduced pressure and theresidue was purified by reverse-phase prep-HPLC to afford 118 (36 mg,38%) as a white solid. MS: [M+H]⁺ 698.3. ¹H NMR (500 MHz, CDCl₃) δ 8.64(d, J=2.0 Hz, 1H), 8.27 (s, 1H), 8.05 (d, J=2.0 Hz, 1H), 7.88 (s, 1H),7.48 (d, J=2.5 Hz, 1H), 7.37 (d, J=5.5 Hz, 1H), 7.30 (dd, J=2.5, 9.0 Hz,1H), 7.11 (dd, J=2.5, 8.5 Hz, 1H), 6.82 (d, J=9.0 Hz, 1H), 4.79-4.73 (m,2H), 4.67-4.61 (m, 2H), 4.31 (s, 2H), 3.77 (t, J=7.0 Hz, 1H), 3.72 (s,3H), 3.21-3.19 (m, 1H), 2.99-2.98 (m, 2H), 2.94-2.91 (m, 1H), 2.88-2.86(m, 2H), 2.78-2.71 (m, 2H), 2.51-2.49 (m, 1H), 1.99-1.96 (m, 6H),0.92-0.90 (m, 6H).

Example 119a2-Bromo-4-fluoro-6-(10-fluoro-1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylAcetate 119a

Example 119a 10-Bromo-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-1-one119a

Into a 250-mL 3-necked round-bottom flask was placed a solution of1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-1-one 104j (9.5 g, 49.94mmol, 1.00 equiv) in N,N-dimethylformamide (100 mL), followed by theaddition of N-bromosuccinimide (9.8 g, 55.06 mmol, 1.10 equiv) inseveral batches at 0° C. The resulting solution was stirred at roomtemperature for 2 h and diluted with 500 mL of water. The precipitatewas filtered and dried in a vacuum oven to afford 9.5 g (71%) of 119a asa light brown solid.

Example 119b10-Fluoro-1H,2H,3H,4H,6H,7H,8H,9H-pyrazino[1,2-a]indol-1-one 119b

Into a 2-L 4-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed a solution of 119a (40 g, 148.62mmol, 1.00 equiv) in tetrahydrofuran (200 mL), followed by the additionof n-BuLi (2.4 M) (218 mL, 3.50 equiv) dropwise with stirring at −78° C.The resulting solution was stirred at −40° C. for 3 h. To this was addeda solution of N-fluorobenzenesulfonimide (98.7 g, 313.33 mmol, 2.10equiv) in tetrahydrofuran (200 mL) dropwise with stirring at −78° C. Theresulting solution was stirred at room temperature for 3 h, quenched bythe addition of 200 mL of water and extracted with 3×500 mL of ethylacetate. The combined organic layers were dried over anhydrous sodiumsulfate and concentrated under vacuum. The crude product (30 g) waspurified by Prep-HPLC with the following conditions (mobile phase, A:0.05% trifluoroacetic acid/water; B: CH₃CN; gradient: 10% B-25% B) toafford 5.05 g (16%) of 119b as a white solid. MS: [M+H]⁺ 209. 1H NMR(300 MHz, CDCl₃) δ 6.16 (br, 1H), 3.90-3.86 (m, 2H), 3.65-3.62 (m, 2H),2.53-2.47 (m, 4H), 1.88-1.80 (m, 2H), 1.77-1.72 (m, 2H).

Example 119c2-Bromo-4-fluoro-6-(10-fluoro-1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylAcetate 119c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (60 mL),2,6-dibromo-4-fluorobenzyl acetate 101C (2.34 g, 7.2 mmol), 119b (500mg, 2.4 mmol), and cesium carbonate (1.6 g, 4.8 mmol). After bubblingnitrogen through the resulting mixture for 30 minutes, Xantphos (140 mg,0.24 mmol) and tris(dibenzylideneacetone)dipalladium(0) (220 mg, 0.24mmol) were added and the reaction mixture was heated at 100° C. for 12h. After this time the reaction was cooled to room temperature andfiltered. The filtrate was partitioned between ethyl acetate (40 mL) andwater (40 mL). The aqueous layer was separated and extracted with ethylacetate (3×70 mL). The combined organic layer was washed with brine (30mL) and dried over sodium sulfate. The drying agent was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresidue was purified by silica-gel column eluting with 3:1 petroleumether/ethyl acetate to afford 119c (632 mg, 58%) as a yellow solid. MS:[M+H]⁺ 453.2

Example 119d(S)-4-Fluoro-2-(10-fluoro-1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)benzylAcetate 119d

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 119c (150 mg, 1.0 eq.,0.33 mmol),(S)-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one113f (160 mg, 1.0 eq., 0.33 mmol), K₃PO₄ (210 mg, 3.0 eq., 0.99 mmol),PdCl₂(dppf) (27.0 mg, 0.10 eq., 0.033 mmol), THF (20 mL), and water (0.1mL). After three cycles of vacuum/argon flush, the mixture was heated atreflux for 2 h. It was then cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure and the resultingresidue was purified by silica-gel column chromatography eluting with40:1 dichloromethane/methanol to afford 119d as a yellow solid (90 mg,37%). MS: [M+H]⁺ 728.3.

Example 119(S)-10-fluoro-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one119

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 119d (90 mg, 1.0 eq., 0.12 mmol), lithiumhydroxide (9.0 mg, 3.0 eq., 0.37 mmol), i-propanol (3 mL), THF (3 mL),and water (2 mL). The mixture was stirred at room temperature for 1 h.It was then filtered and concentrated. The residue was purified byreverse-phase prep-HPLC to afford 119 (42 mg, 49%). MS: [M+H]⁺ 686.3. ¹HNMR (500 MHz, CDCl₃) δ 8.54 (dd, J=2.0 Hz, 9.0, 1H), 7.94-7.93 (m, 1H),7.81 (d, J=4.0 Hz, 1H), 7.45-7.44 (m, 1H), 7.31 (dd, J=3.0, 9.0 Hz, 1H),7.15-7.14 (m, 1H), 6.94 (dd, J=2.0, 9.0 Hz, 1H), 6.81 (d, J=8.5 Hz, 1H),4.71-4.61 (m, 4H), 4.53 (d, J=9.5 Hz, 1H), 4.32-4.31 (m, 2H), 4.15-4.08(m, 3H), 3.89-3.86 (m, 1H), 3.69 (s, 3H), 3.55-3.43 (m, 2H), 3.07 (m,2H), 2.57-2.46 (m, 7H), 2.20-2.16 (m, 1H), 1.88-1.76 (m, 4H), 0.98-096(m, 3H).

Example 120a{2-[5-({5-[(2S)-2-Ethyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl]-4-fluoro-6-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-5-yl}phenyl}methylAcetate 120a

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with(S)-5-bromo-3-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one114e (179 mg, 0.40 mmol),(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 117e (200 mg, 0.40 mmol), Pd(dppf)Cl₂ (29 mg, 0.04 mmol), K₃PO₄(170 mg, 0.8 mmol), sodium acetate (66 mg, 0.8 mmol), acetonitrile (5mL), and water (1.0 mL). After three cycles of vacuum/argon flush, themixture was heated at reflux for 2 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 120aas a yellow solid (120 mg, 41%). MS: [M+H]⁺ 740.3

Example 1203-(3-{5-[54(S)-2-Ethyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridin-3-yl}-5-fluoro-2-hydroxymethyl-phenyl)-6,7,8,9-tetrahydro-3H-benzo[4,5]thieno[2,3-d]pyridazin-4-one120

A mixture of 120a (120 mg, 0.16 mmol) and lithium hydroxide (38 mg, 1.6mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at 30°C. for 1 h. The mixture was evaporated under reduced pressure. Theresulting residue was extracted with ethyl acetate (2×10 mL). Thecombined organic layer was concentrated under reduced pressure and theresidue was purified by reverse-phase prep-HPLC to afford 120 (73 mg,65%) as a white solid. MS: [M+H]⁺ 698.3. ¹H NMR (500 MHz, CDCl₃) δ 8.58(d, J=1.5 Hz, 1H), 8.26 (s, 1H), 7.94 (d, J=2.5 Hz, 1H), 7.80 (s, 1H),7.45 (d, J=2.5 Hz, 1H), 7.30 (d, J=9.0 Hz, 1H), 7.27 (d, J=9.0 Hz, 1H),7.11 (dd, J=2.5, 8.0 Hz, 1H), 6.82 (d, J=9.0 Hz, 1H), 4.72-4.62 (m, 4H),4.31 (s, 2H), 4.01 (bs, 1H), 3.71 (s, 3H), 3.53 (t, J=6.0 Hz, 1H),3.34-3.32 (m, 1H), 3.13 (t, J=6.0 Hz, 2H), 2.99 (t, J=5.0 Hz, 2H), 2.87(t, J=5.0 Hz, 2H), 2.60-2.56 (m, 1H), 2.46-2.44 (m, 2H), 2.36-2.34 (m,1H), 2.01-1.96 (m, 4H), 1.71-1.68 (m, 1H), 1.44-1.36 (m, 1H), 0.83 (t,J=7.5 Hz, 3H).

Example 121a Ethyl 4,5,6,7-Tetrahydro-1H-indole-2-carboxylate 121a

To a mixture of ethyl 3-(2-chlorocyclohex-1-enyl)acrylate (21.4 g, 100mmol) in DMSO (100 mL) was added sodium azide (9.75 g, 150 mmol). Thereaction mixture was heated at 105° C. for 4 h. After cooling to roomtemperature, the mixture was poured into ice water. The resultingprecipitate was collected by filtration to afford 121a (18.0 g, 93.3%).MS-ESI: [M+H]⁺ 194.

Example 121b Ethyl1-(2,2-Diethoxyethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 121b

To a suspension of NaH (1.44 g, 60.2 mmol) in N,N-dimethylformamide(DMF)(30 mL) was slowly added 121a (5.80 g, 30.1 mmol) at 0° C. Theresulting mixture was stirred at room temperature for 0.5 h, followed bythe addition of 2-bromo-1,1-diethoxyethane (11.9 g, 60.2 mmol). Thereaction was heated at 70° C. for 30 h and quenched with water (100 mL).The mixture was then extracted with ethyl acetate (3×100 mL). Thecombined organic phase was concentrated under reduced pressure and theresidue was purified with silica-gel column chromatography eluting with40:1 petroleum ether/ethyl acetate to afford 121b (4.7 g, 51%). MS-ESI:[M-ethanol+H]⁺ 264. ¹H NMR (500 MHz, DMSO-d₆) δ 6.65 (s, 1H), 4.59 (t,J=5.0 Hz, 1H), 4.17-4.16 (m, 4H), 3.59-3.57 (m, 2H), 3.27-3.26 (m, 2H),2.61 (t, J=6.0 Hz, 2H), 2.51 (t, J=6.0 Hz, 2H), 1.73-1.71 (m, 2H),1.63-1.61 (m, 2H), 1.25 (t, J=7.0 Hz, 3H), 1.02 (t, J=7.0 Hz, 6H).

Example 121c1-(2,2-Diethoxyethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylic Acid121c

To a mixture of 121b (4.7 g, 15.2 mmol) in a mixed solvent of ethanol(20 mL), tetrahydrofuran (20 mL), and water (30 mL) was added sodiumhydroxide (3.0 g, 75.0 mmol). The reaction was heated at 75° C. for twodays and concentrated under reduced pressure. The residue was suspendedin water and neutralized with diluted aqueous citric acid solution. Themixture was extracted with ethyl acetate (3×100 mL) and the combinedorganic phase was concentrated under reduced pressure to afford 121c(3.32 g, 78%). MS-ESI: [M-ethanol+H]⁺ 236.

Example 121d1-(2,2-Diethoxyethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxamide 121d

To a mixture of 121c (2.8 g, 10.0 mmol) in N,N-dimethylformamide (30 mL)was added O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) (5.7 g, 15.0 mmol), triethylamine (1.5 g,15.0 mmol), and DMAP (128 mg, 1.0 mmol). The reaction mixture wasstirred at room temperature overnight. Saturated ammonium hydroxide (30mL) was added and the resulting mixture was further stirred for 2 h. Itwas then diluted with water (100 mL) and extracted with ethyl acetate(3×100 mL). The combined organic phase was concentrated under reducedpressure and the residue was purified by silica-gel columnchromatography eluting with petroleum ether/ethyl acetate (6:1 to 3:1)to afford 121d (2.7 g, 96%). MS-ESI: [M-ethanol+H]⁺ 235. ¹H NMR (500MHz, DMSO) δ 7.35 (bs, 1H), 6.70 (bs, 1H), 6.60 (s, 1H), 4.60 (t, J=5.5Hz, 1H), 4.18 (d, J=4.0 Hz, 2H), 3.57-3.56 (m, 2H), 3.25 (m, 2H), 2.57(t, J=6.0 Hz, 2H), 2.40 (t, J=6.0 Hz, 2H), 1.71 (t, J=5.0 Hz, 2H), 1.64(t, J=5.0 Hz, 2H), 1.01 (t, J=7.0 Hz, 6H).

Example 121e 6,7,8,9-Tetrahydropyrazino[1,2-a]indol-1(2H)-one 121e

A mixture of 121d (2.7 g, 9.6 mmol) and acetic acid (10 mL) was heatedat 110° C. for 2 h. The mixture was cooled to room temperature andneutralized with aqueous sodium carbonate solution and extracted withethyl acetate (3×30 mL). The combined organic phase was concentratedunder reduced pressure to afford 121e as a yellow solid (1.6 g, 88%).MS-ESI: [M+H]⁺ 189.3. ¹H NMR (500 MHz, DMSO-d₆) δ 10.28 (s, 1H), 7.02(d, J=5.5 Hz, 1H), 6.63 (s, 1H), 6.52 (pt, J=5.5 Hz, 1H), 2.66 (t, J=6.0Hz, 2H), 2.57 (t, J=6.0 Hz, 2H), 1.83-1.82 (m, 2H), 1.73-1.72 (m, 2H).

Example 121f2-Bromo-4-fluoro-6-(1-oxo-6,7,8,9-tetrahydropyrazino[1,2-a]indol-2(1H)-yl)benzaldehyde121f

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 121e (500 mg, 2.66mmol), 2,6-dibromo-4-fluorobenzaldehyde (1.50 g, 5.32 mmol), andpotassium acetate (521 mg, 5.32 mmol). After bubbling argon through thesuspension for 30 minutes, 4,7-dimethoxy-1,10-phenanthroline (638.4 mg,2.66 mmol) and cuprous iodide (506 mg, 2.66 mmol) were added. The systemwas subjected to three cycles of vacuum/argon flush and then heated at100° C. for 16 h. It was then cooled to room temperature and filtered.The solid was washed with dichloromethane (2×100 ml). The combinedfiltrate was concentrated under reduced pressure and the residue waspurified by silica-gel column chromatography eluting with petroleumether/ethyl acetate (10:1 to 3:1) to afford 121f (510 mg, 49%) as ayellow solid. MS: [M+H]⁺ 389.

Example 121g3-(5-((2S,5R)-2,5-Dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one121g

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with5-bromo-3-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one104e (3.0 g, 6.70 mmol), Pin₂B₂ (8442 mg, 33.5 mmol), Pd₂(dba)₃ (311 mg,0.34 mmol), X-phos (319 mg, 0.67 mmol), potassium acetate (1970 mg, 20.1mmol), and dioxane (50 mL). After three cycles of vacuum/argon flush,the mixture was heated at 60° C. for 16 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure. The resulting residue was washed with 8:1 petroleumether/ethyl acetate (80 mL) to afford 121g as a yellow solid (3 g, 90%).MS: [M+H]⁺ 496.4.

Example 121h2-(5-(5-((2S,5R)-2,5-Dimethyl-4-(oxetan-3-yl)piperazin-1-yl)-pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-6,7,8,9-tetrahydropyrazino[1,2-a]indol-2(1H)-yl)benzaldehyde121h

A 50-mL round-bottomed flask was charged with 121f (194 mg, 0.5 mmol),121g (347.0 mg, 0.7 mmol), potassium acetate (98.0 mg, 1.0 mmol),1,1′-bis(diphenylphosphino)ferrocene-dichloropalladium(II) (20.4 mg,0.025 mmol), water (0.5 mL), and acetonitrile (20 mL). The system wassubjected to 3 cycles of vacuum/argon flush and heated at 100° C. underargon atmosphere and for 3 h. Analysis of the reaction mixture by LCMSshowed little start materials remained. The reaction mixture was cooledto room temperature and filtered. The filtrate was diluted withdichloromethane (50 mL) and water (50 mL). The aqueous layer wasseparated and extracted with dichloromethane (3×20 mL). The combinedorganic layer was dried over Na₂SO₄, filtered, and concentrated underreduced pressure. The dark residue was purified by silica-gel columnchromatography eluting with dichloromethane/methanol (80/1 to 30/1) toafford 121g (182 mg, 54%) as yellow solid. MS: [M+H]⁺ 678.

Example 1212-(3-(5-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6,7,8,9-tetrahydropyrazino[1,2-a]indol-1(2H)-one121

To a solution of 121h (150 mg, 0.22 mmol) in methanol (10 mL) was addedNaBH₄ (41.8 mg, 1.1 mmol) at room temperature. After the reaction wasstirred for 1 h, LCMS indicated the reaction was complete. The mixturewas poured into water (30 mL) and concentrated under reduced pressure.The residue was extracted with dichloromethane (3×30 mL). The combinedorganic layer was washed with brine (30 mL), dried over Na₂SO₄,filtered, and concentrated under reduced pressure. The residue solid waspurified by prep-HPLC to afford 121 (60.3 mg, 40%) as white solid. MS:[M+H]⁺ 680. ¹H NMR (500 MHz, DMSO-d₆) δ 8.63 (d, J=2.0 Hz, 1H), 8.51 (s,1H), 7.90 (d, J=3.0 Hz, 1H), 7.41 (m, 2H), 7.31-7.23 (m, 4H), 6.78 (d,J=1.0 Hz, 2H), 4.80 (m, 1H), 4.55-4.54 (m, 2H), 4.49-4.4.48 (m, 2H),4.28-4.20 (m, 2H), 3.69-3.64 (m, 1H), 3.60 (s, 3H), 3.29-3.27 (m, 2H),2.89-2.88 (m, 1H), 2.75-2.67 (m, 4H), 2.61 (m, 2H), 1.93-1.86 (m, 3H),1.59 (m, 2H), 0.85-0.81 (m, 6H).

Example 122a(S)-3-(5-(2-Ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one122a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with(S)-5-bromo-3-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridine-2-ylamino)-1-methylpyridin-2(1H)-one114e (3219 mg, 7.20 mmol), Pin₂B₂ (9072 mg, 36.0 mmol), Pd₂(dba)₃ (329mg, 0.36 mmol), X-phos (302 mg, 0.72 mmol), potassium acetate (2117 mg,21.6 mmol), and dioxane (50 mL). After three cycles of vacuum/argonflush, the mixture was heated at 60° C. for 16 h. It was then cooled toroom temperature and filtered. The filtrate was concentrated underreduced pressure and the resulting residue was washed with 8:1 petroleumether/ethyl acetate (80 mL) to afford 122a as a yellow solid (3.0 g,84%).

Example 122b(S)-2-(5-(5-(2-Ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-6,7,8,9-tetrahydropyrazino[1,2-a]indol-2(1H)-yl)benzaldehyde122b

A round-bottomed flask was charged with2-bromo-4-fluoro-6-(1-oxo-6,7,8,9-tetrahydropyrazino[1,2-a]indol-2(1H)-yl)benzaldehyde121f (159 mg, 0.41 mmol), 122a (213 mg, 0.43 mmol), PdCl₂(dppf) (29 mg,0.04 mmol), K₃PO₄ (182 mg, 0.86 mmol), sodium acetate (71 mg, 0.86mmol), acetonitrile (15 mL), and water (1.5 mL). After three cycles ofvacuum/argon flush, the mixture was heated at 80° C. for 3 h. It wasthen filtered and the filtrate was evaporated under reduced pressure.The residue was purified by silica-gel column chromatography elutingwith 1:20 methanol/dichloromethane to afford 122b as a red solid (120mg, 43%). MS: [M+H]⁺ 678.3

Example 122(S)-2-(3-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6,7,8,9-tetrahydropyrazino[1,2-a]indol-1(2H)-one122

A mixture of 122b (100 mg, 0.15 mmol), NaBH₄ (22 mg, 0.60), and methanol(10 mL) was stirred at 25° C. for 1 h. It was then quenched with water(5 mL) and concentrated under reduced pressure. The residue wasextracted with dichloromethane (2×10 mL) and the combined organic layerwas concentrated under reduced pressure. The residue was purified byreverse-phase prep-HPLC to afford 122 (32 mg, 31%). MS: [M+H]⁺ 680.3. ¹HNMR (500 MHz, CDCl₃) δ 8.56 (s, 1H), 7.91 (s, 1H), 7.80 (s, 1H), 7.53(s, 1H), 7.28-7.26 (m, 2H), 7.05 (s, 1H), 6.97-6.93 (m, 2H), 6.81 (d,J=8.5 Hz, 1H), 6.45 (d, J=5.5 Hz, 1H), 4.71-4.61 (m, 4H), 4.38 (d,J=12.0 Hz, 1H), 4.36 (d, J=11.5 Hz, 1H), 3.70 (s, 3H), 3.52 (bs, 1H),3.31 (d, J=5.5 Hz, 1H), 3.13-3.10 (m, 2H), 2.75-2.70 (m, 4H), 2.56-2.43(m, 4H), 1.98-1.96 (m, 2H), 1.85-1.84 (m, 2H), 1.39-1.36 (m, 2H), 0.82(t, J=7.0 Hz, 3H).

Example 123a2-(5-(5-(2-Ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-2(1H)-yl)benzylAcetate 123a

A 100-mL single-neck round-bottomed flask was charged with2-bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-2(1H)-yl)benzylacetate 101h (120 mg, 0.27 mmol),(S)-3-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one122a (158.4 mg, 0.32 mmol), Pd(dppf)Cl₂ (24.5 mg, 0.03 mmol), K₃PO₄(114.5 mg, 0.54 mmol), sodium acetate.trihydrate (73.4 mg, 0.54 mmol),water (0.5 mL), and acetonitrile (20 mL). The system was evacuated andrefilled with N₂. The reaction mixture was heated at 100° C. for 2 h. Itwas then cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure and the resulting residue waspurified by silica-gel column chromatography eluting with 25:1dichloromethane/methanol to afford 123a (105 mg, 53%) as a yellow brownsolid. MS: [M+H]⁺ 724.3.

Example 123(S)-2-(3-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1(2H)-one123

A mixture of 123a (105 mg, 0.15 mmol) and lithium hydroxide (36 mg, 1.5mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at 30°C. for 1 h. The mixture was evaporated under reduced pressure and theresidue was extracted with ethyl acetate (2×10 mL). The combined ethylacetate extract was concentrated under reduced pressure and the residuewas purified by reverse-phase prep-HPLC to afford 123 (40 mg, 40%) as apale pink solid. MS: [M+H]⁺ 682.3. ¹H NMR (500 M, CHCl₃) δ 8.55 (s, 1H),7.93 (s, 1H), 7.82 (s, 1H), 7.51 (s, 1H), 7.29 (d, J=3.5 Hz, 1H),7.15-7.12 (m, 1H), 6.94 (dd, J=3.5 Hz, 10.5, 1H), 6.81 (d, J=10.0 Hz,1H), 6.30 (s, 1H), 4.77-4.74 (m, 4H), 4.72-4.70 (m, 1H), 4.57-4.55 (m,1H), 4.29-4.24 (m, 1H), 4.12-4.05 (m, 1H), 3.90-3.78 (m, 4H), 3.70 (s,3H), 3.52-3.50 (m, 1H), 3.32-3.30 (m, 1H), 3.12-3.10 (m, 2H), 3.04-2.83(m, 2H), 2.81 (m, 2H), 2.57-2.50 (m, 1H), 2.43-2.33 (m, 2H), 2.05-2.00(m, 2H), 1.87-1.85 (m, 2H), 1.49-1.35 (m, 2H), 0.81 (t, J=8.5 Hz, 3H).

Example 124a(2-{4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-fluoro-6-[1-methyl-5-({5-[(2R)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl)methylAcetate 124a

A round-bottomed flask equipped with a magnetic stirrer and a refluxcondenser was charged with(R)-5-bromo-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)pyridin-2(1H)-one115f (152 mg, 0.35 mmol),(2-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-fluoro-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 103g (206 mg, 0.415 mmol), PdCl₂(dppf) (28 mg, 0.04 mmol), K₃PO₄(147 mg, 0.69 mmol), sodium acetate (57 mg, 0.69 mmol), acetonitrile (20mL), and water (2 mL). After three cycles of vacuum/argon flush, themixture was heated at 100° C. for 3 h. It was then filtered and thefiltrate was evaporated under reduced pressure. The residue was purifiedon silica-gel column chromatography eluting with 1:20methanol/dichloromethane to afford 124a as red solid (70 mg, 28%). MS:[M+H]⁺ 724.2

Example 1242-(5-Fluoro-2-hydroxymethyl-3-{1-methyl-5-[5-((R)-2-methyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H,6H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1-one124

A mixture of 124a (59 mg, 0.080 mmol), lithium hydroxide (19 mg, 0.80mmol), THF (10 mL), i-propanol (8 mL), and water (10 mL) was stirred atroom temperature for 1.5 h. It was then concentrated under reducedpressure and the residue was extracted with dichloromethane (2×10 mL).The combined dichloromethane extract was concentrated under reducedpressure. The residue was purified with reverse-phase prep-HPLC toafford 124 (43 mg, 79%). MS: [M+H]⁺ 682.3. ¹H NMR (500 MHz, DMSO-d₆) δ8.58-8.57 (m, 1H), 8.41 (s, 1H), 7.84 (d, J=3.0 Hz, 1H), 7.37-7.31 (m,3H), 7.22 (d, J=9.5 Hz, 1H), 7.19-7.16 (m, 1H), 6.50 (s, 1H), 4.87 (d,J=2.0 Hz, 1H), 4.57-4.53 (m, 2H), 4.46 (t, J=6.0 Hz, 1H), 4.41 (t, J=6.0Hz, 1H), 4.31 (d, J=3.0 Hz, 2H), 4.21-4.18 (m, 2H), 4.15-4.10 (m, 1H),3.88-3.85 (m, 1H), 3.67 (d, J=2.0 Hz, 1H), 3.58 (s, 3H), 3.41-3.37 (m,2H), 3.10-3.07 (m, 1H), 2.95-2.91 (m, 1H), 2.56 (d, J=1.5 Hz, 2H), 2.41(s, 2H), 2.32-2.28 (m, 2H), 2.17-2.15 (m, 1H), 1.21 (s, 6H), 0.91 (d,J=6.5 Hz, 3H)

Example 125a tert-Butyl3,3-Dimethyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 125a

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 5-bromo-2-nitropyridine(5.6 g, 28.0 mmol), tert-butyl 3,3-dimethyl-piperazine-1-carboxylate(3.0 g, 14.0 mmol), cesium carbonate (9.1 g, 28 mmol), and 1,4-dioxane(50 mL). After bubbling nitrogen through the resulting solution for 30min, Binap (870 mg, 1.4 mmol) andtris(dibenzylideneacetone)dipalladium(0) (1.2 g, 1.4 mmol) were added.The reaction mixture was subjected to three cycles of vacuum/argon flushand stirred at 120° C. for 24 h. After this time the reaction was cooledto room temperature it was filtered and the filtrate was partitionedbetween ethyl acetate (200 mL) and water (50 mL). The aqueous layer wasseparated and extracted with ethyl acetate (3×50 mL). The combinedorganic layer was washed with brine (50 mL) and dried over sodiumsulfate. The drying agent was removed by filtration and the filtrate wasconcentrated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with 5:1 petroleum ether/ethylacetate to afford 125a (1.27 g, 27%). LCMS: [M+H]⁺ 337.2.

Example 125b tert-Butyl4-(6-Aminopyridin-3-yl)-3,3-dimethylpiperazine-1-carboxylate 125b

A 50-mL round-bottomed flask was purged with nitrogen and charged with125a (1100 mg, 3.2 mmol), 10% palladium on carbon (10% wet, 110 mg), andmethanol (20 mL). It was then evacuated, charged with hydrogen gas, andstirred at room temperature for 5 h. The hydrogen was evacuated andnitrogen was charged into the flask. The catalyst was removed byfiltration through a pad of Celite and the filtrate was concentratedunder reduced pressure to afford 125b (950 mg, 94%). LCMS: [M+H]⁺ 307.3

Example 125c tert-Butyl4-(6-(5-Bromo-1-methyl-2-oxo-1,2-dihydropyridin-3-ylamino)pyridin-3-yl)-3,3-dimethylpiperazine-1-carboxylate 125c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 125b (950 mg, 3.1 mmol),3,5-dibromo-1-methylpyridin-2(1H)-one (1240 mg, 4.6 mmol), 1,4-dioxane(30 mL), and cesium carbonate (2015 mg, 6.2 mmol). After bubblingnitrogen through the resulting solution for 5 min, Xantphos (179 mg,0.31 mmol) and tris(dibenzylideneacetone)dipalladium(0) (283 mg, 0.31mmol) were added. The reaction mixture was subjected to three cycles ofvacuum/argon flush and heated at reflux for 10 h. After this time thereaction was cooled to room temperature and filtered. The filtrate waspartitioned between ethyl acetate (50 mL) and water (10 mL). The aqueouslayer was separated and extracted with ethyl acetate (3×20 mL). Thecombined organic layer was washed with brine (30 mL) and dried oversodium sulfate. The drying agent was removed by filtration and thefiltrate was concentrated under reduced pressure. The residue waspurified by silica-gel column chromatography eluting with 4:1 petroleumether/ethyl acetate to afford 125c (1.21 g, 79%). LCMS: [M+H]⁺ 492.1.

Example 125d5-Bromo-3-(5-(2,2-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one125d

To the solution of 125c (1.19 g, 1.9 mmol) in dichloromethane (20 mL)was added 3M HCl in diethyl ether (15 mL). The reaction mixture wasstirred at room temperature for 4 h. It was then concentrated underreduced pressure to afford 125d (900 mg, 95%). LCMS: [M+H]⁺ 392.1.

Example 125e5-Bromo-3-(5-(2,2-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methylpyridin-2(1H)-one125e

A mixture of 125d (900 mg, 2.3 mmol), oxetan-3-one (497 mg, 6.9 mmol),NaBH₃CN (435 mg, 6.9 mmol), and zinc chloride (311 mg, 2.3 mmol) inmethanol (30 mL) was stirred at 50° C. for 4 hours. It was thenconcentrated under reduced pressure. water (10 mL) was added to theresidue and the mixture was extracted with CHCl₃ 3×50 mL). The combinedorganic layer was concentrated under reduced pressure. The residue waspurified by silica-gel column-chromatography eluting with 50:1dichloromethane/methanol to afford 125e (800 mg, 78%). LCMS: [M+H]⁺448.1. ¹H NMR (500 MHz, CDCl₃) δ 8.65 (d, J=2.0 Hz, 1H), 8.11 (d, J=2.5Hz, 1H), 7.85 (s, 1H), 7.37-7.34 (m, 1H), 6.96 (d, J=2.5 Hz, 1H), 6.72(d, J=8.5 Hz, 1H), 4.69-4.61 (m, 4H), 3.60 (s, 3H), 3.50-3.14 (m, 3H),2.43-2.17 (m, 4H), 1.06 (s, 6H).

Example 125f2-(5-(5-(2,2-Dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylAcetate 125f

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 125e (190 mg, 1.0 eq.,0.42 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetra-methyl-1,3,2-dioxaborolan-2-yl)benzylacetate 115m (405 mg, 2.0 eq., 0.84 mmol), PdCl₂(dppf) (33 mg, 0.10 eq.,0.040 mmol), K₃PO₄ (178 mg, 2.0 eq., 0.84 mmol), sodium acetate (69 mg,2.0 eq., 0.84 mmol), acetonitrile (20 mL), and water (0.1 mL). Afterthree cycles of vacuum/argon flush, the mixture was heated at 90° C. for2 h. It was then cooled to room temperature and filtered. The filtratewas concentrated under reduced pressure and the resulting residue waspurified by silica-gel column chromatography eluting with 50:1dichloromethane/ethanol to afford 125f (90 mg, 29%) as yellow solid. MS:[M+H]⁺ 724.3.

Example 1252-(3-(5-(5-(2,2-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one125

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and was charged with 125f (85 mg, 1 eq., 0.11 mmol), lithiumhydroxide (14 mg, 5 eq., 0.55 mmol), i-propanol (3 mL), THF (3 mL) andwater (2 mL). The mixture was stirred at 30° C. for 1 h. It was thenfiltered and the residue was concentrated under reduced pressure. Theresidue was purified by reverse-phase prep-HPLC to afford 125 (43 mg,57%). MS: [M+H]⁺ 682.4. ¹H NMR (500 MHz, DMSO) δ 8.62 (d, J=2.0 Hz, 1H),8.55 (s, 1H), 7.94 (d, J=2.5 Hz, 1H), 7.41-7.39 (m, 2H), 7.33-7.31 (m,1H), 7.23-7.17 (m, 2H), 6.52 (s, 1H), 4.86 (brs, 1H), 4.54 (t, J=6.5 Hz,2H), 4.42 (t, J=6.0 Hz, 2H), 4.30 (s, 2H), 4.17-4.11 (m, 3H), 3.89-3.86(m, 1H), 3.58 (m, 3H), 3.39-3.36 (m, 1H), 3.08-2.98 (m, 2H), 2.63-2.56(m, 2H), 2.46 (t, J=6.0 Hz, 2H), 2.33-2.12 (m, 4H), 1.80-1.67 (m, 4H),0.96 (s, 6H).

Example 126a2-(5-(5-((2S,5R)-2,5-Dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-2(1H)-yl)benzylAcetate 126a

A 100-mL single-neck round-bottomed flask was charged with2-bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-2(1H)-yl)benzylacetate 101h (347 mg, 0.80 mmol),3-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one121g (792 mg, 1.6 mmol), Pd(dppf)Cl₂ (32.7 mg, 0.040 mmol), K₃PO₄ (340.0mg, 1.6 mmol), sodium acetate trihydrate (217.6 mg, 1.6 mmol), water(0.5 mL), and acetonitrile (50 mL). The system was evacuated andrefilled with N₂. The reaction mixture was heated at 100° C. for 2 h. Itwas then cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure and the resulting residue waspurified by silica-gel column chromatography eluting with 25:1dichloromethane/methanol to afford 126a (200 mg, 34.6%) as a yellowbrown solid. MS: [M+H]⁺ 724.5.

Example 1262-[3-[5-[[5-[(2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-1-methyl-6-oxo-3-pyridyl]-5-fluoro-2-(hydroxymethyl)phenyl]-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1-one126

A mixture of 126a (150 mg, 0.20 mmol) and lithium hydroxide (72 mg, 3.0mmol) in i-propanol/THF (5:3, 8.0 mL) and water (2.0 mL) was stirred at30° C. for 1 h. The mixture was evaporated under reduced pressure andthe residue was extracted with ethyl acetate (2×20 mL). The combinedethyl acetate extract was concentrated under reduced pressure and theresidue was purified by prep-HPLC to afford 126 (45 mg, 33%) as a whitesolid. MS: [M+H]⁺ 682.9. ¹H NMR (500 MHz, CHCl₃) δ 8.60 (dd, J=2, 5 Hz,1H), 8.03 (s, 1H), 7.87 (s, 1H), 7.54 (s, 1H), 7.37-7.35 (m, 1H),7.14-7.12 (m, 1H), 6.96-6.94 (m, 1H), 6.82-6.80 (m, 1H), 6.31 (s, 1H),4.77-4.71 (m, 2H), 4.67-4.62 (m, 2H), 4.58-4.55 (m, 1H), 4.41-4.40 (m,1H), 4.29-4.25 (m, 1H), 4.13-4.09 (m, 1H), 3.91-3.80 (m, 3H), 3.77-3.74(m, 1H), 3.70 (s, 3H), 3.18 (d, J=5.0 Hz, 1H), 3.06-3.01 (m, 1H),2.98-2.90 (m, 2H), 2.83 (m, 2H), 2.77-2.70 (m, 2H), 2.47 (m, 1H),2.06-2.01 (m, 2H), 1.97-1.93 (m, 1H), 1.88-1.87 (m, 2H), 0.91-0.89 (m,6H).

Example 127a Methyl 2-(Hydroxy(pyridin-2-yl)methyl)acrylate 127a

A 250-mL single-neck round-bottomed flask was charged with chloroform(100 mL), picolinaldehyde (10.7 g, 0.10 mol), methyl acrylate (8.60 g,0.10 mol), and 1,4-diazabicyclo[2.2.2]octane (0.560 g, 5.00 mmol). Thereaction mixture stirred at room temperature for 48 h. After this timethe reaction was concentrated under reduced pressure and the residue waspurified by silica-gel column chromatography eluting with 3:1 petroleumether/ethyl acetate to afford 127a as a dark yellow oil (11.6 g, 60%).MS-ESI: (M+H)⁺ 194.2. ¹H NMR (500 MHz, CDCl₃) δ 8.54 (d, J=5.0 Hz, 1H),7.69-7.66 (m, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.22-7.20 (m, 1H), 6.36 (s,1H), 5.97 (s, 1H), 5.62 (s, 1H), 4.85 (s, 1H), 3.74 (s, 3H).

Example 127b Methyl Indolizine-2-carboxylate 127b

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with acetic anhydride (80 mL)and 127a (6.68 g, 34.6 mmol). The reaction mixture was heated at refluxunder nitrogen for 4 h. After this time the reaction was cooled to roomtemperature, poured onto the mixture of ice (100g) and saturated aqueoussodium bicarbonate solution (200 mL), and stirred for 1 h. The resultingsolution was neutralized with saturated aqueous sodium bicarbonate andextracted with methylene chloride (3×200 mL). The combined organicextract was dried over sodium sulfate and concentrated under reducedpressure. The residue was purified by silica-gel column chromatographyeluting with 10:1 petroleum ether/ethyl acetate (10:1) to afford 127b asa white solid (2.1 g, 35%). MS-ESI: (M+H)⁺ 176.2. ¹H NMR (500 MHz,CDCl₃) δ 7.86-7.84 (m, 1H), 7.79 (d, J=1.0 Hz, 1H), 7.36-7.34 (m, 1H),6.82 (s, 1H), 6.70-6.66 (m, 1H), 6.55-6.51 (m, 1H), 3.88 (s, 3H).

Example 127c Methyl 5,6,7,8-Tetrahydroindolizine-2-carboxylate 127c

A 250-mL round-bottomed flask was purged with nitrogen and charged with127b (2.0 g, 11.4 mmol), 10% palladium on carbon (50% wet, 200 mg), andmethanol (50 mL). It was evacuated, charged with hydrogen gas, andstirred under 5 atm hydrogen at room temperature for 8 h. The hydrogenwas then evacuated and nitrogen was charged into the flask. The catalystwas removed by filtration through a pad of CELITE® and the filtrateconcentrated under reduced pressure to afford 127c as a white solid (1.1g, 81%). MS-ESI: [M+H]⁺ 180.3. ¹H NMR (500 MHz, DMSO-d₆) δ 7.25 (d,J=2.0 Hz, 1H), 6.09 (s, 1H), 3.93 (t, J=6.0 Hz, 2H), 3.66 (s, 3H), 2.67(t, J=6.0 Hz, 2H), 1.87-1.83 (m, 2H), 1.75-1.70 (m, 2H).

Example 127d Methyl 3-Formyl-5,6,7,8-tetrahydroindolizine-2-carboxylate127d

A 100-mL round-bottomed flask equipped with a magnetic stirrer waspurged with nitrogen and charged with anhydrous dichloroethane (20 mL)and anhydrous DMF (0.70 mL, 9.0 mmol). To the mixture cooled at 0° C.was added phosphorus oxychloride (0.70 mL, 7.3 mmol) over a period of 2min, while maintaining the reaction temperature between 0 and 10° C. Thecooling bath was removed and the reaction was stirred at roomtemperature for 1 hour. A solution of methyl5,6,7,8-tetrahydroindolizine-2-carboxylate (127c) (1.0 g, 5.6 mmol) inacetonitrile (10 mL) was added and the reaction mixture was stirred atroom temperature for 3 hours. After this time, it was concentrated underreduced pressure. The oily residue was taken up with saturated aqueousNaHCO₃ (20 mL) and extracted with ethyl acetate (3×50 mL). The combinedorganic layer was washed with water (50 mL), dried over Na₂SO₄, andevaporated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with 1:5 ethylacetate/petroleum ether to afford 127d as a white solid (703 mg, 58%).MS-ESI: (M+H)⁺208.3. ¹H NMR (500 MHz, DMSO-d₆) δ 10.14 (s, 1H), 6.40 (s,1H), 4.27 (t, J=6.0 Hz, 2H), 3.78 (s, 3H), 2.78 (t, J=6.0 Hz, 2H),1.94-1.85 (m, 2H), 1.78-1.69 (m, 2H).

Example 127e 6,7,8,9-Tetrahydropyridazino[4,5-b]indolizin-1(2H)-one 127e

A 100-mL single-neck round-bottomed flask equipped with a refluxcondenser was charged with methyl3-formyl-5,6,7,8-tetrahydroindolizine-2-carboxylate (127d) (600 mg, 2.9mmol) and hydrazinium hydroxide (20 mL). The reaction mixture was heatedat 100° C. for 4 hours. After this time the reaction was cooled to roomtemperature and filtered to afford 127e as a yellow solid (413 mg, 75%).MS-ESI: (M+H)⁺ 190.1. ¹H NMR (500 MHz, DMSO-d₆) δ 12.17 (s, 1H), 8.24(s, 1H), 6.33 (s, 1H), 4.16 (t, J=6.0 Hz, 2H), 2.88 (t, J=6.5 Hz, 2H),2.00-1.96 (m, 2H), 1.84-1.79 (m, 2H).

Example 127f2-Bromo-4-fluoro-6-(1-oxo-6,7,8,9-tetrahydropyridazino[4,5-b]indolizin-2(1H)-yl)benzaldehyde127f

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 127e (450 mg, 2.4 mmol),2,6-dibromo-4-fluorobenzaldehyde (2.0 g, 7.2 mmol), cesium carbonate(1.6 g, 4.8 mmol), and 1,4-dioxane (50 mL). After bubbling nitrogenthrough the resulting mixture for 10 minutes, copper(I) iodide (450 mg,2.4 mmol) and 4,7-dimethoxy-1,10-phenanthroline (571 mg, 2.4 mmol) wereadded, and the reaction mixture was heated at 90° C. for 12 h. Afterthis time the reaction was cooled to room temperature and filtered. Thefiltrate was partitioned between methylene chloride (40 mL) and water(40 mL). The aqueous layer was separated and extracted with methylenechloride (3×30 mL). The combined organic layer was washed with brine (50mL) and dried over sodium sulfate. The drying agent was removed byfiltration and the filtrate was concentrated under reduced pressure. Theresidue was purified by silica-gel column chromatography eluting with1:2 ethyl acetate/petroleum ether to afford 127f as a brown solid (251mg, 31%). MS-ESI: (M+H)⁺ 390.0.

Example 127g4-Fluoro-2-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)-6-(1-oxo-6,7,8,9-tetrahydropyridazino[4,5-b]indolizin-2(1H)-yl)benzaldehyde127g

A 100-mL round-bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with 127f (125.0 mg, 0.32 mmol),1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetra-methyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one113f (155.0 mg, 0.32 mmol), sodium acetate (53.0 mg, 0.64 mmol), K₃PO₄(135.7.0 mg, 0.64 mmol), PdCl₂(dppf) (50.0 mg, 0.06 mmol), acetonitrile(25 mL), and water (1 mL). The system was subjected to 3 cycles ofvacuum/argon flush and heated at 100° C. for 3 hours. It was thenevaporated under reduced pressure and the residue was purified bysilica-gel column chromatography eluting with 30:1 methylenechloride/methanol to afforded 127g compound (108 mg, 51%) as a brownsolid. MS: [M+H]⁺ 665.4.

Example 1272-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]phenyl]-6,7,8,9-tetrahydropyridazino[4,5-b]indolizin-1-one127

A solution of 127g (100.0 mg, 0.15 mmol) in methanol (20 mL) was addedNaBH₄ (17.0 mg, 0.45 mmol). The mixture was stirred at room temperaturefor 2 h. It was then quenched with water (1 mL) and the mixture wasevaporated under reduced pressure. The residue was purified byreverse-phase prep-HPLC to afford 127 (56 mg, yield 56%) as a whitesolid. MS: [M+H]⁺ 667.4. ¹H NMR (500 MHz, DMSO-d₆) δ 8.58 (d, J=2.0,1H), 8.47 (s, 1H), 8.40 (s, 1H), 7.85 (d, J=2.5, 1H), 7.39 (d, J=2.0,1H), 7.37-7.34 (m, 1H), 7.30-7.28 (m, 1H), 7.25-7.22 (m, 2H), 6.48 (s,1H), 4.57-4.53 (m, 2H), 4.46 (m, 2H), 4.41 (m, 1H), 4.25 (m, 2H), 4.20(s, 2H), 3.69-3.64 (m, 1H), 3.58 (s, 3H), 3.41-3.36 (m, 1H), 3.10-3.07(m, 1H), 2.96-2.92 (m, 3H), 2.54-2.50 (m, 1H), 2.35-2.28 (m, 2H), 2.18(m, 1H), 2.04-2.00 (m, 2H), 1.87-1.82 (m, 2H), 0.92 (d, J=6.0, 3H).

Example 128a2-Bromo-6-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-fluorobenzaldehyde128a

A 100-mL round-bottomed flask equipped with a reflux condenser wascharged with4,4-dimethyl-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-9-one103e (1.0 g, 4.90 mmol, 1.0 eq.), 2-bromo-6-chloro-4-fluorobenzaldehyde(2.76 g, 9.8 mmol, 2.0 eq.), Pd₂(dba)₃ (224 mg, 0.24 mmol, 0.050 eq.),Xantphos (283 mg, 0.49 mmol, 0.10 eq.), potassium acetate (1.44 g, 14.7mmol, 3.0 eq.), and 1,4-dioxane (50 mL). The system was evacuated andrefilled with N₂. The reaction mixture was heated at 80° C. for 5 h.After this time the reaction was cooled to room temperature andfiltered. The filtrate was concentrated under reduced pressure and theresidue was purified on silica-gel column eluting with 80:1dichloromethane/methanol to afford 128a as a yellow solid (992 mg, 50%).MS: [M+H]⁺ 405.1

Example 128b2-{4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-fluoro-6-[1-methyl-5-({5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]benzaldehyde128b

A 50-mL round-bottomed flask equipped with a magnetic stirrer and areflux condenser was charged with 128a (303 mg, 0.75 mmol),1-methyl-3-({5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxa-borolan-2-yl)-1,2-dihydropyridin-2-one113f (385 mg, 0.80 mmol), Pd(dppf)Cl₂ (68.6 mg, 0.075 mmol), potassiumacetate (147 mg, 1.50 mmol), K₃PO₄ (327 mg, 1.50 mmol), acetonitrile (15mL), and water (6 drops). After three cycles of vacuum/argon flush, themixture was heated at 100° C. for 2 h. After this time the reaction wascooled to room temperature. It was then filtered and the filtrate wasevaporated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with 15:1 ethylacetate/methanol to afford 128b (382 mg, 77%) as a black solid. MS-ESI:[M+H]⁺ 680.3

Example 1282-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-4-fluoro-6-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]benzoicacid 128

To a mixture of 128b (190 mg, 0.28 mmol), tert-butyl alcohol (7 mL), anddichloromethane (0.5 mL) was added 2-methyl-2-butene (9.0 mL, 107 mmol).An aqueous solution (2 mL) of NaClO₂ (53 mg, 0.59 mmol) and NaH₂PO₄.2water (135.7 mg, 0.87 mmol) was added drop-wise at −10° C. The mixturewas stirred at −10° C. for 1 h. It was then treated with water (20 mL)and extracted with ethyl acetate (4×50 mL). The combined organic extractwas dried over MgSO₄, filtered, and evaporated under reduced pressure.The residue was purified with reverse-phase prep-HPLC to afford 128 (33mg, 17%) as a pale yellow solid. MS-ESI: [M+H]⁺ 696.2. ¹H NMR (500 MHz,DMSO-d₆) δ 13.19-13.17 (m, 1H), 8.60 (s, 1H), 8.36 (s, 1H), 7.86 (d,J=2.5 Hz, 1H), 7.37-7.35 (m, 2H), 7.24-7.22 (m, 3H), 6.46 (s, 1H),4.57-4.54 (m, 2H), 4.48-4.47 (m, 1H), 4.42-4.41 (m, 1H), 4.10-4.09 (m,2H), 3.95-3.92 (m, 1H), 3.68-3.67 (m, 1H), 3.56 (s, 3H), 3.42-3.39 (m,2H), 3.09-3.07 (m, 1H), 2.96-2.94 (m, 1H), 2.92 (s, 3H), 2.41-2.40 (m,2H), 2.35-2.31 (m, 2H), 2.20-2.17 (m, 1H), 1.21-1.20 (m, 6H), 0.93 (d,J=6.5 Hz, 3H).

Example 129a2-Bromo-6-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-fluorobenzoicAcid 129a

To a mixture of2-bromo-6-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-fluorobenzaldehyde128a (810 mg, 2.0 mmol), tert-butyl alcohol (50 mL), and dichloromethane(3 mL) was added 2-methyl-2-butene (22 mL, 262 mmol). An aqueoussolution (20 mL) of NaClO₂ (1.8 g, 20.0 mmol) and NaH₂PO₄ dihydrate (2.2g, 14.0 mmol) was added dropwise at 0° C. The mixture was stirred at 0°C. for 1 h. It was then treated with water (30 mL) and extracted withethyl acetate (4×90 mL). The combined organic extract was dried overMgSO₄ and concentrated under reduced pressure to afford 129a (930 mg,84%) as a yellow solid. MS-ESI: [M+H]⁺ 421.1

Example 129b2-Bromo-6-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-fluoro-N-methylbenzamide 129b

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with DMF (8 mL), 129a (160 mg, 0.38 mmol), HATU (505mg, 1.33 mmol), DMAP (46 mg, 0.38 mmol), and triethylamine (1.0 mL). Themixture was heated at 25° C. for 0.5 h. Then MeNH₂.HCl (266 mg, 3.8mmol) was added and resulting mixture was stirred at 25° C. for 2.5 h.After this time the reaction was cooled to room temperature. It was thenfiltered and the filtrate was evaporated under reduced pressure. Theresidue was purified by prep-TLC developing with 1:20methanol/dichloromethane to afford 129b (116 mg, 70%) as a black solid.MS-ESI: [M+H]⁺ 434.0

Example 1292-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-4-fluoro-N-methyl-6-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]benzamide129

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 129b (116 mg, 0.27mmol),1-methyl-3-({5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2-dihydropyridin-2-one113f (260 mg, 0.54 mmol), Pd(dppf)Cl₂ (26 mg, 0.030 mmol), potassiumacetate (53 mg, 0.54 mmol), K₃PO₄ (117 mg, 0.54 mmol), acetonitrile (5mL), and water (0.50 mL). After three cycles of vacuum/argon flush, themixture was heated at reflux for 2 h. After this time the reaction wascooled to room temperature. It was then filtered and the filtrate wasevaporated under reduced pressure. The residue was purified withreverse-phase prep-HPLC to afford 129 (80 mg, 42%) as a pale yellowsolid. MS-ESI: [M+H]⁺ 709.5. ¹H NMR (500 MHz, DMSO-d₆) δ 8.69 (d, J=2.0Hz, 1H), 8.39 (s, 1H), 8.11 (d, J=4.5 Hz, 1H), 7.89 (d, J=2.5 Hz, 1H),7.38-7.36 (m, 1H), 7.32-7.30 (m, 2H), 7.28-7.26 (m, 1H), 7.23-7.21 (m,1H), 6.47 (s, 1H), 4.58-4.54 (m, 2H), 4.48-4.46 (m, 1H), 4.43-4.41 (m,1H), 4.05-3.91 (m, 4H), 3.67-3.66 (m, 1H), 3.57 (s, 3H), 3.41-3.38 (m,1H), 3.10-3.08 (m, 1H), 2.97-2.94 (m, 1H), 2.55-2.54 (m, 3H), 2.48-2.47(m, 3H), 2.40-2.39 (m, 2H), 2.36-2.28 (m, 2H), 2.22-2.19 (m, 1H),1.21-1.20 (m, 6H), 0.93 (d, J=6.5 Hz, 3H).

Example 130a2-Bromo-6-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-5-yl}benzaldehyde130a

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (20 mL),8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-6-one117c (618 mg, 3.0 mmol), 2,6-dibromobenzaldehyde (1980 mg, 7.5 mmol),CuBr (215 mg, 1.5 mmol), sarcosine (267 mg, 3.0 mmol), and K₂CO₃ (828mg, 6.0 mmol). After three cycles of vacuum/argon flush, the mixture washeated at 100° C. for 16 h. After this time the reaction was cooled toroom temperature. It was then filtered and the filtrate was evaporatedunder reduced pressure. The residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 130a(702 mg, 60%) as a yellow solid. MS: [M+H]⁺ 389.0

Example 130b2-[1-Methyl-5-({5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxopyridin-3-yl]-6-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-5-yl}benzaldehyde130b

A sealed tube equipped with a magnetic stirrer was charged with 130a(160 mg, 0.40 mmol),(S)-1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-ylboronicacid 113f (160 mg, 0.40 mmol), Pd(dppf)Cl₂ (32 mg, 0.040 mmol), sodiumacetate (66 mg, 0.80 mmol), K₃PO₄ (170 mg, 0.80 mmol), and acetonitrile(6 mL). After three cycles of vacuum/argon flush, the mixture was heatedat 100° C. for 2 h. It was then filtered and the filtrate was evaporatedunder reduced pressure. The residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 130b(123 mg, 46%) as a yellow solid. LCMS: [M+H]⁺ 664.3

Example 1303-[2-(hydroxymethyl)-3-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]phenyl]-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-4-one130

At 0° C., to a solution of 130b (120 mg, 0.18 mmol) in methanol (5 mL)was added sodium borohydride (20 mg, 0.54 mmol). The reaction wasstirred for 30 minutes. It was then quenched with water (1 mL) andconcentrated under reduced pressure. The residue was purified byreverse-phase prep-HPLC to afford 130 (70 mg, 59%). LCMS: [M+H]⁺ 666.3.¹H NMR (500 MHz, CDCl₃) δ 8.62 (d, J=2.0 Hz, 1H), 8.26 (s, 1H), 7.98 (s,1H), 7.84 (s, 1H), 7.56-7.54 (m, 2H), 7.42 (d, J=2.5 Hz, 1H), 7.37 (dd,J=2.0, 7.0 Hz, 1H), 7.31 (d, J=8.0 Hz, 1H), 6.83 (d, J=8.5 Hz, 1H),4.72-4.66 (m, 4H), 4.36 (d, J=5.0 Hz, 2H), 4.05 (t, J=5.5 Hz, 1H), 3.72(s, 3H), 3.55-3.54 (m, 1H), 3.46-3.45 (m, 1H), 3.08-3.06 (m, 2H), 2.99(t, J=5.0 Hz, 2H), 2.87 (t, J=5.0 Hz, 2H), 2.60-2.59 (m, 1H), 2.49-2.48(m, 2H), 2.20-2.19 (m, 1H), 2.02-1.96 (m, 4H), 0.99 (d, J=6.0 Hz, 3H).

Example 131a 3,3-Dimethylcyclopentanone 131a

To a suspension of CuI (81.0 g, 420 mmol) in anhydrous ethyl ether (500mL) cooled to 0° C. was added a solution of methyllithium in ethyl ether(430 mL, 860 mmol, 2.0M) over a period of 30 minutes. See FIG. 15. Themixture was stirred at 0° C. for 2 h. To the above mixture was added3-methylcyclopent-2-enone (33.6 g, 350 mmol) dropwise over a period of 1h at 0° C. The resulting mixture was stirred at 0° C. for another 2 h.It was then quenched with saturated NH₄Cl (300 mL) and filtered. Thefiltrate was extracted with ethyl ether (2×200 mL). The combined organiclayer was dried over anhydrous Mg₂SO₄ and filtered. The filtrate wasevaporated under reduced pressure to afford 131a as a colorless oil (28g, 71%). ¹H NMR (500 MHz, DMSO-d₆) δ 2.31 (t, J=8.0 Hz, 2H), 2.05 (s,2H), 1.79 (t, J=8.0 Hz, 2H), 1.12 (s, 6H).

Example 131b 2-Chloro-4,4-dimethylcyclopent-1-enecarbaldehyde 131b

To a solution of DMF (18.3 g, 250 mmol) in dichloromethane (300 mL)cooled at 0° C. was added POCl₃ (40.5 g, 250 mmol) over a period of 10minutes. See FIG. 15. The mixture was stirred at 20° C. for 1 h. To theabove mixture was added 131a (28.0 g, 250 mmol) dropwise over a periodof 20 minutes. The resulting mixture was heated at reflux for 20 h. Thereaction mixture was cooled to room temperature and poured into asolution of sodium acetate (60 g) in ice-water (400 g). The mixture wasextracted with dichloromethane (2×300 mL). The combined organic layerwas washed with water (2×200 mL), dried over anhydrous Mg₂SO₄ andfiltered. The filtrate was evaporated under reduced pressure to afford131b as a colorless oil (33.0 g, crude). ¹H NMR (500 MHz, DMSO-d₆) δ9.99 (s, 1H), 2.62 (d, J=2.0 Hz, 2H), 2.38 (d, J=2.0 Hz, 2H), 1.15 (s,6H).

Example 131c Ethyl5,5-Dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxylate 131c

To a solution of 131b (33.0 g, crude) in dichloromethane (400 mL) andtriethylamine (60 g, 600 mmol) was added ethyl 2-mercaptoacetate (19.2g, 160 mmol). See FIG. 15. The reaction mixture was heated at reflux for6 h. It was then concentrated under reduced pressure. The residue wasdissolved in ethanol (400 mL) and triethylamine (60 g, 600 mmol). Themixture was heated at reflux for 12 h. It was concentrated again underreduced pressure and the residue was purified by silica-gel columnchromatography eluting with 40:1 petroleum ether/ethyl acetate to 131cas a yellow solid (18.0 g, 32%, over two steps). MS-ESI: [M+H]⁺ 225.3.¹H NMR (500 MHz, DMSO-d₆) δ 7.49 (s, 1H), 4.32 (q, J=7.0 Hz, 2H), 2.72(s, 2H), 2.56 (s, 2H), 1.35 (t, J=7.0 Hz, 3H), 1.22 (s, 6H).

Example 131d5,5-Dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxylic Acid131d

To a solution of 131c (16.0 g, 71.0 mmol) in propan-2-ol (200 mL),tetrahydrofuran (200 mL), and water (200 mL) was added lithium hydroxide(6.82 g, 284 mmol). See FIG. 15. The reaction mixture was heated at 65°C. for 5 h. The organic solvents were removed under reduced pressure.The pH of the residue was adjusted to 1.0 with hydrochloride acid (12M).The precipitate was collected by filtration and dried in vacuo to afford131d (12.0 g, 86%) as a white solid. MS-ESI: [M+H]⁺ 196.9

Example 131eN-tert-Butyl-5,5-dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxamide131e

A suspension of 131d (12.0 g, 61.0 mmol) in SOCl₂ (80 mL) was heated at65° C. for 2 h. The reaction mixture was concentrated under reducedpressure. See FIG. 15. The residue was diluted with dichloromethane (20mL), which was added to the solution of 2-methylpropan-2-amine (4.45 g,61.0 mmol) and triethylamine (18.0 g, 180 mmol) in dichloromethane (180mL). The resulting mixture was stirred for 16 h and diluted withdichloromethane (200 mL). It was washed with water (3×50 mL), dried overanhydrous Mg₂SO₄, filtered, and evaporated under reduced pressure toafford 131e (15.0 g, 97%) as a yellow solid. MS-ESI: [M+H]⁺ 252.0

Example 131fN-tert-Butyl-3-formyl-5,5-dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxamide131f

To a solution of 131e (1.5 g, 6.0 mmol) in anhydrous THF (60 mL) cooledto −70° C. was added the solution of n-butyllithium (10.0 mL, 25 mmol,2.5 Min hexane) over a period of five minutes. See FIG. 15. It wasstirred at −70° C. for 6 h. DMF (1.3 g, 18.0 mmol) was added over aperiod of five minutes and the result mixture was stirred at roomtemperature overnight. It was then quenched with saturated NH₄Cl (40 mL)and concentrated under reduced pressure. The residue was extracted withethyl acetate (2×30 mL). The combined organic layer was dried overanhydrous Mg₂SO₄ and filtered. The filtrate was evaporated under reducedpressure to afford 131f as a yellow solid (1.34 g, 80%). MS-ESI: [M+H]⁺280.3

Example 131gN-tert-Butyl-3-(hydrazonomethyl)-5,5-dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxamide131g

To a solution of 85% aqueous hydrazine (10 mL) in THF (180 mL) was added131f (5.6 g, 20.0 mmol) in anhydrous THF (20 mL) over a period of 5minutes. See FIG. 15. It was stirred at 20° C. for 3 h. The reactionmixture was concentrated under reduced pressure to afford 131g as ablack solid (6.0 g, yield: 95%, purity: 95%). MS-ESI: [M+H]⁺ 294.0

Example 131h4,4-Dimethyl-7-thia-10,11-diazatricyclo[6.4.0.0^(2,6)]dodeca-1(8),2(6),11-trien-9-one131h

A solution of 131g (3.8 g, 13.0 mmol) in 30% H₂SO₄ (100 mL) was heatedat reflux for 16 h. See FIG. 15. The reaction mixture was cooled to roomtemperature and extracted with dichloromethane (3×200 mL). The combinedorganic layer was concentrated under reduced pressure and the residuewas purified by silica-gel column chromatography eluting with 100:1dichloromethane/methanol to afford 131h as a yellow solid (1.72 g, 60%).MS-ESI: [M+H]⁺ 221.0

Example 131i2-Bromo-6-{4,4-dimethyl-9-oxo-7-thia-10,11-diazatricyclo[6.4.0.0^(2,6)]dodeca-1(8),2(6),11-trien-10-yl}-4-fluorobenzaldehyde131i

A 100-mL round bottom flask equipped with a magnetic stirrer and areflux condenser was charged with 131h (330 mg, 1.5 mmol),2,6-dibromo-4-fluorobenzaldehyde (1.26 g, 4.5 mmol), CuBr (113 mg, 0.8mmol), sarcosine (142 mg, 1.6 mmol), K₂CO₃ (420 mg, 3.0 mmol), anddioxane (20 mL). See FIG. 15. After three cycles of vacuum/argon flush,the mixture was heated at 95° C. for 15 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography 5:1 eluting with petroleum ether/ethyl acetate to 131i asa white solid (380 mg, 60%). MS-ESI: [M+H]⁺ 420.6

Example 131j2-{4,4-Dimethyl-9-oxo-7-thia-10,11-diazatricyclo[6.4.0.0^(2,6)]dodeca-1(8),2(6),11-trien-10-yl}-4-fluoro-6-[1-methyl-5-({5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridin-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]benzaldehyde131j

A 50-mL round bottom flask equipped with a magnetic stirrer and a refluxcondenser was charged with 131i (421 mg, 1.0 mmol),(S)-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2(1H)-one113f (580 mg, 1.2 mmol), Pd(dppf)Cl₂ (59 mg, 0.080 mmol),K₃PO₄.trihydrate (360 mg, 1.6 mmol), water (6 drops), andtetrahydrofuran (20 mL). After three cycles of vacuum/argon flush, themixture was heated at reflux for 6 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the residue was washed with 1:1 petroleum ether/ethylacetate (20 mL) to afford 131j as a white solid (556 mg, 80%). MS-ESI:[M+H]⁺ 696.3

Example 1313-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]phenyl]-7,7-dimethyl-6,8-dihydrocyclopenta[3,4]thieno[1,3-d]pyridazin-4-one131

To a solution of 131j (520 mg, 0.75 mmol) in methanol (10 mL) was addedsodium borohydride (110 mg, 3.0 mmol) at 20° C. The reaction mixture wasstirred for 30 minutes and quenched with water (3 mL). It was thenconcentrated under reduced pressure and the residue was purified withreverse phase prep-HPLC to afford 131 (340 mg, 65%). MS-ESI: [M+H]⁺698.3. ¹H NMR (500 MHz, DMSO-d₆) δ 8.55 (d, J=2.5 Hz, 1H), 8.46 (s, 1H),8.41 (s, 1H), 7.85 (d, J=2.5 Hz, 1H), 7.40-7.30 (m, 4H), 7.23 (d, J=9.0Hz, 1H), 4.60 (t, J=5.0 Hz, 1H), 4.57-4.53 (m, 2H), 4.46 (t, J=6.0 Hz,1H), 4.41 (t, J=6.0 Hz, 1H), 4.30-4.29 (m, 2H), 3.68-3.65 (m, 1H), 3.58(s, 3H), 3.40-3.38 (m, 1H), 3.10-3.07 (m, 1H), 2.95-2.94 (m, 1H),2.91-2.89 (m, 2H), 2.80-2.78 (m, 2H), 2.54-2.52 (m, 1H), 2.34-2.30 (m,2H), 2.20-2.16 (m, 1H), 1.27 (s, 6H), 0.93 (d, J=6.5 Hz, 3H).

Example 901 Biochemical Btk Assay

A generalized procedure for a standard biochemical Btk Kinase Assay thatcan be used to test Formula I compounds is as follows. A master mixminus Btk enzyme is prepared containing 1× Cell Signaling kinase buffer(25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mMdithiothreitol, 0.1 mM Na₃VO₄, 10 mM MgCl₂), 0.5 μM Promega PTKBiotinylated peptide substrate 2, and 0.01% BSA. A master mix plus Btkenzyme is prepared containing 1× Cell Signaling kinase buffer, 0.5 μMPTK Biotinylated peptide substrate 2, 0.01% BSA, and 100 ng/well (0.06mU/well) Btk enzyme. Btk enzyme is prepared as follows: full lengthhuman wildtype Btk (accession number NM-000061) with a C-terminal V5 and6×His tag was subcloned into pFastBac vector for making baculoviruscarrying this epitope-tagged Btk. Generation of baculovirus is donebased on Invitrogen's instructions detailed in its published protocol“Bac-to-Bac Baculovirus Expression Systems” (Cat. Nos. 10359-016 and10608-016). Passage 3 virus is used to infect Sf9 cells to overexpressthe recombinant Btk protein. The Btk protein is then purified tohomogeneity using Ni-NTA column. The purity of the final proteinpreparation is greater than 95% based on the sensitive Sypro-Rubystaining A solution of 200 μM ATP is prepared in water and adjusted topH7.4 with 1N NaOH. A quantity of 1.25 μL of compounds in 5% DMSO istransferred to a 96-well ½ area Costar polystyrene plate. Compounds aretested singly and with an 11-point dose-responsive curve (startingconcentration is 10 μM; 1:2 dilution). A quantity of 18.75 μL of mastermix minus enzyme (as a negative control) and master mix plus enzyme istransferred to appropriate wells in 96-well ½ area costar polystyreneplate. 5 μL of 200 μM ATP is added to that mixture in the 96-well ½ areaCostar polystyrene plate for final ATP concentration of 40 μM. Thereaction is allowed to incubate for 1 hour at room temperature. Thereaction is stopped with Perkin Elmer 1× detection buffer containing 30mM EDTA, 20 nM SA-APC, and 1 nM PT66 Ab. The plate is read usingtime-resolved fluorescence with a Perkin Elmer Envision using excitationfilter 330 nm, emission filter 665 nm, and 2^(nd) emission filter 615nm. IC₅₀ values are subsequently calculated. Alternatively, theLanthascreen assay can be used to evaluate Btk activity throughquantification of its phosphorylated peptide product. The FRET(Fluorescence Resonance Energy Transfer) that occurs between thefluorescein on the peptide product and the terbium on the detectionantibody decreases with the addition of inhibitors of Btk that reducethe phosphorylation of the peptide. In a final reaction volume of 25 uL,Btk (h) (0.1 ng/25 ul reaction) is incubated with 50 mM Hepes pH 7.5, 10mM MgCl₂, 2 mM MnCl₂, 2 mM DTT, 0.2 mM NaVO4, 0.01% BSA, and 0.4 uMfluorescein poly-GAT. The reaction is initiated by the addition of ATPto 25 uM (Km of ATP). After incubation for 60 minutes at roomtemperature, the reaction is stopped by the addition of a finalconcentration of 2 nM Tb-PY20 detection antibody in 60 mM EDTA for 30minutes at room temperature. Detection is determined on a Perkin ElmerEnvision with 340 nM excitation and emission at 495 nm and 520 nm.Exemplary Btk inhibition IC50 values are in Tables 1, 2, and 3.

Example 902 Ramos Cell Btk Assay

Another generalized procedure for a standard cellular Btk Kinase Assaythat can be used to test Formula I compounds is as follows. Ramos cellsare incubated at a density of 0.5×10⁷ cells/ml in the presence of testcompound for 1 hr at 37° C. Cells are then stimulated by incubating with10 μg/ml anti-human IgM F(ab)₂ for 5 minutes at 37° C. Cells arepelleted, lysed, and a protein assay is performed on the cleared lysate.Equal protein amounts of each sample are subject to SDS-PAGE and westernblotting with either anti-phosphoBtk(Tyr223) antibody (Cell SignalingTechnology #3531; Epitomics, cat. #2207-1) or phosphoBtk(Tyr551)antibody (BD Transduction Labs #558034) to assess Btkautophosphorylation or an anti-Btk antibody (BD Transduction Labs#611116) to control for total amounts of Btk in each lysate.

Example 903 B-Cell Proliferation Assay

A generalized procedure for a standard cellular B-cell proliferationassay that can be used to test Formula I compounds is as follows.B-cells are purified from spleens of 8-16 week old Balb/c mice using aB-cell isolation kit (Miltenyi Biotech, Cat #130-090-862). Testingcompounds are diluted in 0.25% DMSO and incubated with 2.5×10⁵ purifiedmouse splenic B-cells for 30 min prior to addition of 10 μg/ml of ananti-mouse IgM antibody (Southern Biotechnology Associates Cat #1022-01)in a final volume of 100 μl. Following 24 hr incubation, 1 μCi³H-thymidine is added and plates are incubated an additional 36 hr priorto harvest using the manufacturer's protocol for SPA[³H] thymidineuptake assay system (Amersham Biosciences # RPNQ 0130). SPA-bead basedfluorescence is counted in a microbeta counter (Wallace Triplex 1450,Perkin Elmer).

Example 904 T Cell Proliferation Assay

A generalized procedure for a standard T cell proliferation assay thatcan be used to test Formula I compounds is as follows. T cells arepurified from spleens of 8-16 week old Balb/c mice using a Pan T cellisolation kit (Miltenyi Biotech, Cat #130-090-861). Testing compoundsare diluted in 0.25% DMSO and incubated with 2.5×10⁵ purified mousesplenic T cells in a final volume of 100 μl in flat clear bottom platesprecoated for 90 min at 37° C. with 10 μg/ml each of anti-CD3 (BD#553057) and anti-CD28 (BD #553294) antibodies. Following 24 hrincubation, 1 μCi ³H-thymidine is added and plates incubated anadditional 36 hr prior to harvest using the manufacturer's protocol forSPA[³H] thymidine uptake assay system (Amersham Biosciences # RPNQ0130). SPA-bead based fluorescence was counted in a microbeta counter(Wallace Triplex 1450, Perkin Elmer).

Example 905 CD86 Inhibition Assay

A generalized procedure for a standard assay for the inhibition of Bcell activity that can be used to test Formula I compounds is asfollows. Total mouse splenocytes are purified from spleens of 8-16 weekold Balb/c mice by red blood cell lysis (BD Pharmingen #555899). Testingcompounds are diluted to 0.5% DMSO and incubated with 1.25×10⁶splenocytes in a final volume of 200 μl in flat clear bottom plates(Falcon 353072) for 60 min at 37° C. Cells are then stimulated with theaddition of 15 μg/ml IgM (Jackson ImmunoResearch 115-006-020), andincubated for 24 hr at 37° C., 5% CO₂. Following the 24 hr incubation,cells are transferred to conical bottom clear 96-well plates andpelleted by centrifugation at 1200×g×5 min. Cells are preblocked byCD16/CD32 (BD Pharmingen #553142), followed by triple staining withCD19-FITC (BD Pharmingen #553785), CD86-PE (BD Pharmingen #553692), and7AAD (BD Pharmingen #51-68981E). Cells are sorted on a BD FACSCaliburand gated on the CD19⁺/7AAD⁻ population. The levels of CD86 surfaceexpression on the gated population is measured versus test compoundconcentration.

Example 906 B-ALL Cell Survival Assay

The following is a procedure for a standard B-ALL (acute lymphoblasticleukemia) cell survival study using an XTT readout to measure the numberof viable cells. This assay can be used to test Formula I compounds fortheir ability to inhibit the survival of B-ALL cells in culture. Onehuman B-cell acute lymphoblastic leukemia line that can be used isSUP-B15, a human Pre-B-cell ALL line that is available from the ATCC.

SUP-B15 pre-B-ALL cells are plated in multiple 96-well microtiter platesin 100 μA of Iscove's media+20% FBS at a concentration of 5×10⁵cells/ml. Test compounds are then added with a final conc. of 0.4% DMSO.Cells are incubated at 37° C. with 5% CO₂ for up to 3 days. After 3 dayscells are split 1:3 into fresh 96-well plates containing the testcompound and allowed to grow up to an additional 3 days. After each 24 hperiod, 50 ul of an XTT solution is added to one of the replicate96-well plates and absorbance readings are taken at 2, 4 and 20 hoursfollowing manufacturer's directions. The reading taken with an OD forDMSO only treated cells within the linear range of the assay (0.5-1.5)is then taken and the percentage of viable cells in the compound treatedwells are measured versus the DMSO only treated cells.

Example 907 CD69 Whole Blood Assay

Human blood is obtained from healthy volunteers, with the followingrestrictions: 1 week drug-free, non-smokers. Blood (approximately 20 mlsto test 8 compounds) is collected by venipuncture into Vacutainer®(Becton, Dickinson and Co.) tubes with sodium heparin.

Solutions of Formula I compounds at 10 mM in DMSO are diluted 1:10 in100% DMSO, then are diluted by three-fold serial dilutions in 100% DMSOfor a ten point dose-response curve. The compounds are further diluted1:10 in PBS and then an aliquot of 5.5 μl of each compound is added induplicate to a 2 ml 96-well plate; 5.5 μl of 10% DMSO in PBS is added ascontrol and no-stimulus wells. Human whole blood—HWB (100 μl) is addedto each well. After mixing the plates are incubated at 37° C., 5% CO₂,100% humidity for 30 minutes. Goat F(ab′)₂ anti-human IgM (10 μl of a500 μg/ml solution, 50 μg/ml final) is added to each well (except theno-stimulus wells) with mixing and the plates are incubated for anadditional 20 hours. At the end of the 20 hour incubation, samples areincubated with fluorescent labeled antibodies for 30 minutes, at 37° C.,5% CO₂, 100% humidity. Include induced control, unstained and singlestains for compensation adjustments and initial voltage settings.Samples are then lysed with PharM Lyse™ (BD Biosciences Pharmingen)according to the manufacturer's instructions. Samples are thentransferred to a 96 well plate suitable to be run on the BD BiosciencesHTS 96 well system on the LSRII machine. Data acquired and MeanFluorescence Intensity values were obtained using BD Biosciences DIVASoftware. Results are initially analyzed by FACS analysis software (FlowJo). The inhibitory concentrations (IC50, IC70, IC90, etc.) for testcompounds is defined as the concentration which decreases by, forexample 50%, the percent positive of CD69 cells that are also CD20positive stimulated by anti-IgM (average of 8 control wells, aftersubtraction of the average of 8 wells for the no-stimulus background).The IC70 values are calculated by Prism version 5, using a nonlinearregression curve fit and are shown in Tables 1 and 2.

Example 908 In Vitro Cell Proliferation Assay

Efficacy of Formula I compounds are measured by a cell proliferationassay employing the following protocol (Mendoza et al (2002) Cancer Res.62:5485-5488). The CellTiter-Glo® Luminescent Cell Viability Assay,including reagents and protocol are commercially available (PromegaCorp., Madison, Wis., Technical Bulletin TB288). The assay assesses theability of compounds to enter cells and inhibit cell proliferation. Theassay principle is based on the determination of the number of viablecells present by quantitating the ATP present in a homogenous assaywhere addition of the Cell-Titer Glo reagent results in cell lysis andgeneration of a luminescent signal through the luciferase reaction. Theluminescent signal is proportional to the amount of ATP present.

A panel of B-cell lymphoma cell lines (BJAB, SUDHL-4, TMD8, OCI-Ly10,OCI-Ly3, WSU-DLCL2) are plated into 384-well plate in normal growthmedium, and serially diluted BTK inhibitors or DMSO alone were added toeach well. Cell viability is assessed after 96 hour incubation byCellTiter-Glo® (Promega). Data may be presented as Relative cellviability in BTK inhibitor-treated cells relative to DMSO-treatedcontrol cells. Data points are the mean of 4 replicates at each doselevel. Error bars represent SD from the mean.

Procedure: Day 1—Seed Cell Plates (384-well black, clear bottom,microclear, TC plates with lid from Falcon #353962), Harvest cells, Seedcells at 1000 cells per 54 μl per well into 384 well Cell Plates for 3days assay. Cell Culture Medium: RPMI or DMEM high glucose, 10% FetalBovine Serum, 2 mM L-Glutamine, P/S. Incubate 0/N at 37° C., 5% CO2.

Day 2—Add Drug to Cells, Compound Dilution, DMSO Plates (serial 1:2 for9 points), Add 20 μl compounds at 10 mM in the 2nd column of 96 wellplate. Perform serial 1:2 across the plate (10 μl+20 μl 100% DMSO) for atotal of 9 points using Precision. Media Plates 96-well conical bottompolypropylene plates from Nunc (cat. #249946) (1:50 dilution) Add 147 μlof Media into all wells. Transfer 3 μl of DMSO+compound from each wellin the DMSO Plate to each corresponding well on Media Plate usingRapidplate.

Drug Addition to Cells, Cell Plate (1:10 dilution), Add 6 μl ofmedia+compound directly to cells (54 μl of media on the cells already).Incubate 3 days at 37 C, 5% CO2 in an incubator that will not be openedoften.

Day 5—Develop Plates, Thaw Cell Titer Glo Buffer at room temperature.Remove Cell Plates from 37° C. and equilibrate to room temperature. forabout 30 minutes. Add Cell Titer Glo Buffer to Cell Titer Glo Substrate(bottle to bottle). Add 30 μl Cell Titer Glo Reagent (Promega cat. #G7572) to each well of cells. Place on plate shaker for about 30minutes. Read luminescence on Analyst HT Plate Reader (half second perwell).

Cell viability assays and combination assays: Cells were seeded at1000-2000 cells/well in 384-well plates for 16 h. On day two, nineserial 1:2 compound dilutions are made in DMSO in a 96 well plate. Thecompounds are further diluted into growth media using a Rapidplate robot(Zymark Corp., Hopkinton, Mass.). The diluted compounds are then addedto quadruplicate wells in 384-well cell plates and incubated at 37° C.and 5% CO2. After 4 days, relative numbers of viable cells are measuredby luminescence using Cell-Titer Glo (Promega) according to themanufacturer's instructions and read on a Wallac Multilabel Reader(PerkinElmer, Foster City). EC50 values are calculated using Prism® 4.0software (GraphPad, San Diego). Formula I compounds and chemotherapeuticagents are added simultaneously or separated by 4 hours (one before theother) in all assays.

An additional exemplary in vitro cell proliferation assay includes thefollowing steps:

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells inmedium is deposited in each well of a 384-well, opaque-walled plate.

2. Control wells are prepared containing medium and without cells.

3. The compound is added to the experimental wells and incubated for 3-5days.

4. The plates are equilibrated to room temperature for approximately 30minutes.

5. A volume of CellTiter-Glo Reagent equal to the volume of cell culturemedium present in each well is added.

6. The contents are mixed for 2 minutes on an orbital shaker to inducecell lysis.

7. The plate is incubated at room temperature for 10 minutes tostabilize the luminescence signal.

8. Luminescence is recorded and reported in graphs as RLU=relativeluminescence units.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. Accordingly, all suitablemodifications and equivalents may be considered to fall within the scopeof the invention as defined by the claims that follow. The disclosuresof all patent and scientific literature cited herein are expresslyincorporated in their entirety by reference.

We claim:
 1. A compound selected from Formula I:

or stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof, wherein: R¹, R² and R³ are independently selected from H, F,Cl, —NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OH, and C₁-C₃alkyl; R⁴ is selected from H, F, Cl, CN, —CH₂OH, —CH(CH₃)OH, —C(CH₃)₂OH,—CH(CF₃)OH, —CH₂F, —CHF₂, —CH₂CHF₂, —CF₃, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHC(O)CH₃, —OH, —OCH₃, —OCH₂CH₃,—OCH₂CH₂OH, cyclopropyl, cyclopropylmethyl, 1-hydroxycyclopropyl,imidazolyl, pyrazolyl, 3-hydroxy-oxetan-3-yl, oxetan-3-yl, andazetidin-1-yl; R⁵ is selected from —CH₃, and —CH₂CH₃; n is 1, 2, 3, or4; R⁶ is selected from H, —CH₃, —CH₂CH₃, —CH₂CH₂OH, —NH₂, and —OH; R⁷ isselected from the structures:

where the wavy line indicates the site of attachment; R⁸ is selectedfrom —CH₃, —S(O)₂CH₃, cyclopropyl, azetidin-3-yl, oxetan-3-yl, andmorpholin-4-yl; X¹ is CR⁹ or N, where R⁹ is selected from H, F, Cl,—CH₃, —CH₂CH₃, —CH₂CH₂OH, —NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃,and —OCH₂CH₂OH; X² is CR¹⁰ or N, where R¹⁰ is selected from H, —CH₃,—CH₂CH₃, and —CH₂CH₂OH; and Y¹ and Y² are independently selected from CHand N, where Y¹ and Y² are not each N.
 2. The compound of claim 1wherein X¹ is CR⁹, and R⁹ is H.
 3. The compound of claim 1 wherein X¹ isN.
 4. The compound of claim 1 wherein R⁴ is —CH₂OH.
 5. The compound ofclaim 4 wherein R² is F.
 6. The compound of claim 5 wherein R¹ and R³are H.
 7. The compound of claim 1 wherein R⁶ is CH₃.
 8. The compound ofclaim 1 selected from(S)-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1(2H)-one:(S)-5-[5-fluoro-2-(hydroxymethyl)-3(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl]-8-thia-4,5-diazatricyclo[7.4.0.02,7]-trideca-1(9),2(7),3-trien-6-one;(2S)-10-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-({5-[2-methyl-4-(oxetan-3-yl)piperazin-1-yl]pyridine-2-yl}amino)-6-oxo-1,6-dihydropyridin-3-yl]phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.02,6]dodeca-2(6),7-dien-9-one;2-(3-(5-(5-((2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one;(S)-2-(3-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one;(S)-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one;(S)-2-(3-(5-(5-(3,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one;(R)-2-(3-(5-(5-(3,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-α]indol-1(2H)-one;(R)-2-(3-(5-(5-(2,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl-5-fluoro-2-(hydroxymethyl)phenyl-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one;(S)-2-(3-(5-(5-(2,4-dimethylpiperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one;(S)-7,7-difluoro-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one;2-(3-{5-[5-((S)-2-Ethyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-1-methyl-6-oxo-1,6-dihydropyridin-3-yl}-5-fluoro-2-hydroxymethyl-phenyl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H,6H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1-one;(R)-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one;2-(3-{5-[5-((2S,5R)-2,5-Dimethyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridin-3-yl}-5-fluoro-2-hydroxymethyl-phenyl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H,6H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1-one;3-(5-Fluoro-2-hydroxymethyl-3-{1-methyl-5-[5-((R)-2-methyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-6,7,8,9-tetrahydro-3H-benzo[4,5]thieno[2,3-d]pyridazin-4-one;3-(3-{5-[5-((2S,5R)-2,5-Dimethyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridin-3-yl}-5-fluoro-2-hydroxymethyl-phenyl)-6,7,8,9-tetrahydro-3H-benzo[4,5]thieno[2,3-d]pyridazin-4-one;(S)-1-fluoro-2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one;3-(3-{5-[5-((S)-2-Ethyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-1-methyl-6-oxo-1,6-dihydro-pyridin-3-yl}-5-fluoro-2-hydroxymethyl-phenyl)-6,7,8,9-tetrahydro-3H-benzo[4,5]thieno[2,3-d]pyridazin-4-one;2-(3-(5-(5-((2S,5R)-2,5-dimethyl-4(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6,7,8,9-tetrahydropyrazino[1,2-a]indol-1(2H)-one;(S)-2-(3-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6,7,8,9-tetrahydropyrazino[1,2-a]indol-1(2H)-one;(S)-2-(3-(5-(5-(2-ethyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1(2H)-one;2-(5-Fluoro-2-hydroxymethyl-3-{1-methyl-5-[5-((R)-2-methyl-4-oxetan-3-yl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-7,7-dimethyl-3,4,7,8-tetrahydro-2H,6H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1-one;and2-(3-(5-(5-(2,2-dimethyl-4(oxetan-3-yl)piperazin-1-ylpyridin-2-ylamino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one.9. The compound of claim 1 selected from2-[3-[5-[[5-[(2S,5R)-2,5-dimethyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-1-methyl-6-oxo-3-pyridyl]-5-fluoro-2-(hydroxymethyl)phenyl]-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1-one;2-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]phenyl]-6,7,8,9-tetrahydropyridazino[4,5-b]indolizin-1-one;2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-4-fluoro-6-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]benzoicacid;2-(7,7-dimethyl-4-oxo-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-3-yl)-4-fluoro-N-methyl-6-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]benzamide;3-[2-(hydroxymethyl)-3-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]phenyl]-6,7,8,9-tetrahydrobenzothiopheno[2,3-d]pyridazin-4-one;and3-[5-fluoro-2-(hydroxymethyl)-3-[1-methyl-5-[[5-[(2S)-2-methyl-4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-6-oxo-3-pyridyl]phenyl]-7,7-dimethyl-6,8-dihydrocyclopenta[3,4]thieno[1,3-d]pyridazin-4-one.10. A pharmaceutical composition comprised of a compound of claim 1 anda pharmaceutically acceptable carrier, glidant, diluent, or excipient.11. The pharmaceutical composition according to claim 10, furthercomprising a therapeutic agent.
 12. A process for making apharmaceutical composition which comprises combining a compound of claim1 with a pharmaceutically acceptable carrier.
 13. A method of treatingan immune disorder which comprises administering a therapeuticallyeffective amount of the pharmaceutical composition of claim 10 to apatient with the immune disorder.
 14. The method of claim 13 wherein theimmune disorder is rheumatoid arthritis.
 15. The method of claim 13further comprising administering an additional therapeutic agentselected from an anti-inflammatory agent, an immunomodulatory agent,chemotherapeutic agent, a neurotropic factor, an agent for treatingcardiovascular disease, an agent for treating liver disease, ananti-viral agent, an agent for treating blood disorders, an agent fortreating diabetes, and an agent for treating immunodeficiency disorders.16. A kit for treating an immune disorder, comprising: a) apharmaceutical composition of claim 10; and b) instructions for use.